ADAMTS13 genes and proteins and variants, and therapeutic compositions and methods of utilizing the same

ABSTRACT

The present invention relates to a disintegrin and metalloproteinase containing thrombospondin 1-like domains (ADAMTS) and in particular to a novel ADAMTS13 protease and to nucleic acids encoding ADAMTS13 proteases. The present invention encompasses both native and recombinant wild-type forms of ADAMTS13, as well as mutant and variant forms including fragments, some of which posses altered characteristics relative to the wild-type ADAMTS13. The present invention also relates to methods of using ADAMTS13, including for treatment of TTP. The present invention also relates to methods for screening for the presence of TTP. The present invention further relates to methods for developing anticoagulant drugs based upon ADAMTS13.

The present application is a continuation of U.S. patent applicationSer. No. 11/342,461, filed Jan. 30, 2006, issued as U.S. Pat. No.7,517,522 on Apr. 14, 2009, which is a divisional application of U.S.patent application Ser. No. 10/222,334, filed Aug. 16, 2002, issued asU.S. Pat. No. 7,037,658 on May 2, 2006, which claims priority to U.S.Provisional Application Ser. No. 60/312,834, filed Aug. 16, 2001, eachof which is hereby incorporated by reference in its entirety.

The present application was funded with government support under grantnumbers RO1 HL39693 and RO1 HL62131 from the National Institutes ofHealth (NIH). The government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to a disintegrin and metalloproteinasecontaining thrombospondin 1-like domains (ADAMTS), and in particular toa novel ADAMTS13 protease and to nucleic acids encoding ADAMTS13proteases, and to methods of using the same.

BACKGROUND OF THE INVENTION

Thrombotic Thrombocytopenic Purpura (TTP) is a disorder of the bloodcharacterized by low platelets, low red blood cell count (caused bypremature breakdown of the cells), and neurological abnormalities. Thesharp drop in the number of red blood cells and platelets in the bloodis associated with severe problems affecting the kidneys and brain,along with fever and bleeding. Purpura refers to the characteristicbleeding that occurs beneath the skin, or in mucus membranes, whichproduces bruises, or a red rash-like appearance; the bleeding can becatastrophic. The neurological symptoms associated with this diseaseinclude headaches, confusion, speech changes, and alterations inconsciousness, which vary from lethargy to coma; other symptoms includedevelopment of kidney abnormalities. These symptoms can be very severe,and fatal.

Although TTP-like disorders have been associated with variousmedications, bone marrow transplantation, pregnancy, HIV infection, andautoimmune disease, most cases appear sporadically, without an obviousprecipitating factor. This disease is seen most commonly in adults from20 to 50 years old, with women affected slightly more often than men. Inmost TTP patients, the onset of the disease occurs in otherwise healthyindividuals, and there is no history of a similar condition in otherfamily members. However, in a smaller set of individuals, there isevidence suggesting that the condition may be inherited. This evidenceis rare reported cases of familial TTP, where the disease begins earlyin life or sometime shortly after birth, with multiple recurrences andthus a chronic relapsing course; other family members may also beaffected. The disease strikes about 4 out of every 100,000 people.

Current treatment consists of infusion of fresh frozen plasma with orwithout plasma exchange or plasmapheresis. In plasmapheresis, blood iswithdrawn from the patient as for a blood donation. Then the plasmaportion of the blood is removed by passing the blood through a cellseparator. The cells are saved, reconstituted with a plasma substitute,and returned to the patient as a blood transfusion. In TTP, thistreatment is repeated daily until blood tests show improvement. Peoplewho do not respond to this treatment, or who have frequent recurrences,may require removal of the spleen.

Prior to the development of modern treatment protocols, fatality duringan acute episode of TTP was greater than 90% (Rock et al. (1991) N.Engl. J. Med. 325, 393-397; George (2000) Blood 96, 1223-1229).Plasmapheresis has improved the outcome of this disease so that now 80to 90% of patients recover completely; however, fatalities still occur.Although most incidents of the disease are acute, when relapses occur,the disease can become chronic. Despite marked improvement in treatmentoutcome, the molecular pathogenesis of TTP is still unknown and thespecific plasma factor(s) responsible for the acute onset of thisdisease, or recovery following treatment, remains to be identified.Because the cause is unknown, there is no way to prevent the disease.

Thus, what is needed are improved methods to treat the disease, todecrease fatality and to decrease the appearance and/or severity of theconsequent debilitating symptoms associated with the disease. What isalso needed is a method to determine the susceptibility of individualsto the disease, in efforts to prevent the appearance and/or severity ofsymptoms. What is also needed is a method to identify those individualsfor whom the disease appears to be genetic.

SUMMARY OF THE INVENTION

Thus, it is an object of the present invention to provide methods todetermine the susceptibility of individuals to TPP, and to identifythose individuals for whom the disease appears to be genetic. It is afurther object of the present invention to provide improved methods totreat TPP.

These objectives and others are met by the present invention, which insome embodiments provides a method of identifying subjects at risk ofdeveloping TTP disease comprising: providing nucleic acid from asubject, wherein the nucleic acid comprises a ADAMTS13 gene; anddetecting the presence or absence of one or more variations in theADAMTS13 gene. In other embodiments, the method further comprises thestep of determining if the subject is at risk of developing TTP diseasebased on the presence or absence of the one or more variations. In yetother embodiments, in the method of the present invention the variationis a single nucleotide polymorphism, or the variation causes aframeshift mutation in ADAMTS13, or the variation causes a splicemutation in ADAMTS13, or the variation causes a nonconservative aminoacid substitution ADAMTS13; preferably, the variation is selected fromthe group consisting of the mutations shown in Table 1. In someembodiments, in the method of the present invention, the detecting stepis accomplished by hybridization analysis. In further embodiments, thedetecting step comprises comparing the sequence of the nucleic acid tothe sequence of a wild-type ADAMTS13 nucleic acid.

The present invention also provides a method of identifying subjects atrisk of developing TTP disease comprising: providing a blood sample froma subject, wherein the blood sample comprises an ADAMTS13 protease; anddetecting the presence or absence of one or more variants of theADAMTS13 protease. In some embodiments, the detecting step isaccomplished by an antibody assay.

The present invention also provides a kit for determining if a subjectis at risk of developing TTP disease comprising a detection assay,wherein the detection assay is capable of specifically detecting avariant ADAMTS13 allele. In some embodiments, the detection assaycomprises a nucleic acid probe that hybridizes under stringentconditions to a nucleic acid sequence comprising at least one mutationselected from the group consisting of the mutations shown in Table 1.

The invention further provides a kit for determining if a subject is atrisk of developing TTP disease comprising a detection assay, wherein thedetection assay is capable of specifically detecting a variant ADAMTS13protease. In some embodiments, the detection assay comprises an antibodycapable of binding to an ADAMTS13 protease selected from the groupconsisting of wild-type proteases and proteases comprising at least oneamino acid mutation shown in Table 1.

The invention also provides an isolated nucleic acid comprising asequence encoding a polypeptide selected from the group consisting ofSEQ ID NOs: 2 and 4 and variants of SEQ ID NO:2 as shown in Tables 1 and2. In some embodiments, the sequence is operably linked to aheterologous promoter. In further embodiments, the invention provides avector comprising the isolated sequence. In yet further embodiments, theinvention provides a host cell comprising the vector. In someembodiments, the host cell is selected from the group consisting ofanimal and plant cells; in other embodiments, the host cell is locatedin an organism.

The invention also provides an isolated nucleic acid sequence comprisinga sequence selected from the group consisting of SEQ ID NOs: 1 and 3 andvariants of SEQ ID NO:1 as shown in Tables 1 and 2. In some embodiments,the invention provides a computer readable medium encoding arepresentation of a nucleic acid sequence.

The invention also provides an isolated polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 2 and 4and variants of SEQ ID NO:2 as shown in Tables 1 and 2.

The invention also provides a method of identifying subjects at risk ofcarrying an allele for TTP disease comprising: providing nucleic acidfrom a subject, wherein the nucleic acid comprises a ADAMTS 13 gene; anddetecting the presence or absence of one or more variations in theADAMTS13 gene. In other embodiments, the method of the present inventionfurther comprises a step of determining if the subject is at risk ofcarrying TTP disease based on the presence or absence of the one or morevariations.

The present invention also provides an isolated nucleic acid comprisinga sequence encoding a polypeptide CUB domain of ADAMTS13; preferably,the nucleic acid comprises SEQ ID NO: 5. The present invention alsoprovides an isolated polypeptide comprising a CUB domain of ADAMTS13;preferably, the polypeptide comprises SEQ ID NO: 6.

The present invention also provides a method of treating a patient withTTP disease, comprising administering a therapeutically effective amountof ADAMTS13 protease such that the symptoms of the disease arealleviated, wherein the ADAMTS13 protease is selected from the groupconsisting: recombinant ADAMTS13; synthetic ADAMTS13; mutants, variants,fragments, and fusions of recombinant ADAMTS13; and mutants, variants,fragments, and fusions of synthetic ADAMTS13.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the pedigrees used for linkage analysis. VWF-cleavingprotease levels (in U/ml) are indicated beneath the symbol for eachindividual. Affected individuals are indicated by solid symbols andcarriers by dotted symbols. A total of 17 markers as described inExample 1 were used for haplotype analysis. Only select markers areshown. Chromosomes carrying affected alleles are framed, whereas normalchromosomes are not marked. Areas where recombination cannot bedefinitively assigned are indicated by shading. Only recombinationevents between affected and unaffected chromosomes are shown. Inferredgenotypes are indicated in parentheses. Genotypes of unknown phase areindicated by square brackets. Recombination events in individuals AIII3and BII6 place the responsible gene below marker GL2-1 and arecombination event in individual AIII2 places the gene above markerD9S1818.

FIG. 2 shows blood plasma VWF-cleaving protease levels. Panel a showslevels for all individuals shown in FIG. 1, as well as additionalmembers of family A. Panel b shows levels for 61 normal controlindividuals. Affected individuals are indicated by circles, obligatecarriers (parents of affected individuals) by triangles, otherindividuals at-risk for inheriting an affected allele by diamonds, andadditional not at-risk members of family A by hexagons. Normal controlsare shown as triangles. Levels for at-risk individuals (diamonds inpanel a fall into a bimodal distribution, with one peak ranging from0.45-0.68 U/ml, consistent with carriers and the other from 0.90-1.17U/ml, indistinguishable from the normal distribution shown in panel b.

FIG. 3 shows the identification of the ADAMTS13 gene. Panel a shows aphysical map of chromosome 9 in the interval surrounding marker D9S164.The 2.3 Mb nonrecombinant interval identified in FIG. 1 is locatedbetween the markers that designate this interval, which are shown inlarger and bold type. Sequence gaps in public genomic draft assembly aredenoted by black bars. Transcripts localized to this interval aredepicted by black and hatched bars; the different patterns are usedsolely to make it easier to see the individual transcripts in areaswhere they are spaced closely together. The predicted gene C9ORF8 isindicated with an asterisk. The reference bar represents 1 Mb. Panel bshows the intron-exon of an ADAMTS13 gene and the domain structure ofthe encoded ADAMTS13 protein. The coding regions are indicated by graybars and the 5′ and 3′ untranslated regions are indicated by patternedbars. Intron sizes are not drawn to scale. Exon 1 of C9ORF8 overlapswith a cluster of EST sequences (Unigene cluster Hs. 149184), initiallyinterpreted as predicting a large 5′ untranslated region. A segment ofputative C9ORF8 coding sequence was used to identify 2 partial humanfetal liver cDNA clones, which were extended in both the 5′ and 3′direction by RT-PCR and RACE. The assembled cDNA sequence corrected anerror in the predicted boundaries of C9ORF8 exon 2, resulting in acontinuous open reading frame including two exons upstream of the 5′ ESTcluster, 3 new exons within the predicted intron 10 of C9ORF8 and 6additional downstream exons encompassing a second hypothetical gene inthis region, DKFZp434C2322 (Unigene cluster Hs.131433). Analysis ofRT-PCR and cDNA sequences identified an alternatively spliced variant ofexon 17 using both alternate donor and acceptor splice sites; thealternatively spliced exon pieces are indicated by black bars. Mutationsare depicted underneath the corresponding exons, with trianglesrepresenting missense mutations and squares representing frameshift andsplice mutations. The reference bars represents 200 nucleotides. Thepredicted domain structure of ADAMTS13 is shown at the bottom of panelb. The predicted signal peptide is indicated as “SP,” the shortpropeptide is indicated as “pro,” the metalloproteinase domain isindicated by “metalloprotease,” the disintegrin domain is indicated by“disintegrin,” and TSP1 domains are indicated as ovals. The locations ofthe zinc-binding catalytic consensus sequence within themetalloproteinase domain and the cysteine rich region within the spacerdomain are also indicated. The CUB domain (indicated as “CUB”) has notbeen identified in other ADAMTS family members. The reference barrepresents 50 amino acids. Panel c shows the domain structure ofADAMTS13, with the locations of mutations indicated. Missense mutationsidentified in TPP patients are indicated by arrows. The asteriskindicates an additional mutant identified in a TPP family.

FIG. 4 shows the results of Northern and RT-PCR analysis of ADAMTS13.Panel a shows a human Northern blot hybridized with a probe spanningexons 11-13 and part of exon 14. An ˜4.7 kb message can be seenspecifically in the liver and a truncated, ˜2.3 kb message is faintlyvisible in placenta. Panel b shows a panel of cDNAs derived from humantissues screened by PCR for the presence of exons 11-14. Strong signalswere seen in the liver and ovary, with weak expression also evident inkidney pancreas, spleen, thymus prostate, testis, intestine andperipheral blood leukocytes. No expression was detected in heart, brain,placenta, lung or muscle.

FIG. 5 shows the nucleotide sequence of an ADAMTS13 cDNA which encodes along form of ADAMTS13 (SEQ ID NO: 1). This sequence includes ambiguitycodes for all single nucleotide polymorphisms. The IUPAC ambiguity codesare as follows:

M=A or C

R=A or G

W=A or T

S=C or G

Y=C or T

K=G or T

FIG. 6 shows the amino acid sequence of a long form of an ADAMTS13 (SEQID NO:2) encoded by the nucleotide sequence of FIG. 5. This sequencecontains one of the two possible amino acids for regions where SingleNucleotide Polymorphisms (SNPs) change an amino acid; the SNPs andencoded amino acids are shown in Table 2.

FIG. 7 shows the nucleotide sequence of an ADAMTS13 cDNA which encodes ashort form of an ADAMTS13 (SEQ ID NO:3). This sequence includesambiguity codes for all Single Nucleotide Polymorphisms (SNPs). TheIUPAC ambiguity codes are as indicated for FIG. 5.

FIG. 8 shows the amino acid sequence of a short form of ADAMTS13 (SEQ IDNO:4). This sequence contains one of the two possible amino acids forregions where Single Nucleotide Polymorphisms (SNPs) change an aminoacid; the SNPs and encoded amino acids are shown in Table 2.

FIG. 9 shows the amino acid sequence (panel a, SEQ ID NO:5) and thenucleotide sequence (panel b, SEQ ID NO:6) of an ADAMTS13 CUB domain.

FIG. 10 shows the nucleotide sequence of an ADAMTS13 gene which encodesa wild-type ADAMTS13 (SEQ ID NO:7). This sequence includes ambiguitycodes for some Single Nucleotide Polymorphisms (SNPs). The IUPACambiguity codes are as indicated for FIG. 5.

FIG. 11 shows the VWF-cleaving protease activity of ADAMTS13 mutants.VWF-cleaving protease activity was measure in conditioned media ofCHO-Tag cells transfected with wild-type (WT) and mutant ADAMTS13constructs. Activities are represented as the percentage of the activityof wild-type recombinant ADAMTS13.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases as used herein are defined below:

The term “thrombotic thrombocytopenic purpura” or “TTP” refers to adisease characterized by intravascular destruction of erythrocytes andconsumption of blood platelets resulting in anemia and thrombocytopenia.Diffuse platelet rich microthrombi are observed in multiple organs, withthe major extravascular manifestations including fever, and variabledegrees of neurologic and renal dysfunction. Purpura refers to thecharacteristic bleeding that occurs beneath the skin, or in mucusmembranes, which produces bruises, or a red rash-like appearance.

The term “ADAMTS13” refers to a protein encoded by ADAMTS13, a generesponsible for familial TTP. ADAMTS13 has been identified as a uniquemember of the metalloproteinase gene family, ADAM (A Disintegrin AndMetalloproteinase), whose members are membrane-anchored proteases withdiverse functions. ADAMTS family members are distinguished from ADAMs bythe presence of one or more thrombospondin 1-like (TSP1) domain(s) atthe C-terminus and the absence of the EGF repeat, transmembrane domainand cytoplasmic tail typically observed in ADAM metalloproteinases. Itis contemplated that ADAMTS13 possesses VWF (von Wildebrandt factor)cleaving protease activity.

The terms “protein” and “polypeptide” refer to compounds comprisingamino acids joined via peptide bonds and are used interchangeably. A“protein” or “polypeptide” encoded by a gene is not limited to the aminoacid sequence encoded by the gene, but includes post-translationalmodifications of the protein.

Where the term “amino acid sequence” is recited herein to refer to anamino acid sequence of a protein molecule, “amino acid sequence” andlike terms, such as “polypeptide” or “protein” are not meant to limitthe amino acid sequence to the complete, native amino acid sequenceassociated with the recited protein molecule. Furthermore, an “aminoacid sequence” can be deduced from the nucleic acid sequence encodingthe protein.

The term “portion” when used in reference to a protein (as in “a portionof a given protein”) refers to fragments of that protein. The fragmentsmay range in size from four amino acid residues to the entire aminosequence minus one amino acid.

The term “chimera” when used in reference to a polypeptide refers to theexpression product of two or more coding sequences obtained fromdifferent genes, that have been cloned together and that, aftertranslation, act as a single polypeptide sequence. Chimeric polypeptidesare also referred to as “hybrid” polypeptides. The coding sequencesincludes those obtained from the same or from different species oforganisms.

The term “fusion” when used in reference to a polypeptide refers to achimeric protein containing a protein of interest joined to an exogenousprotein fragment (the fusion partner). The fusion partner may servevarious functions, including enhancement of solubility of thepolypeptide of interest, as well as providing an “affinity tag” to allowpurification of the recombinant fusion polypeptide from a host cell orfrom a supernatant or from both. If desired, the fusion partner may beremoved from the protein of interest after or during purification.

The term “homolog” or “homologous” when used in reference to apolypeptide refers to a high degree of sequence identity between twopolypeptides, or to a high degree of similarity between thethree-dimensional structure or to a high degree of similarity betweenthe active site and the mechanism of action. In a preferred embodiment,a homolog has a greater than 60% sequence identity, and more preferablygreater than 75% sequence identity, and still more preferably greaterthan 90% sequence identity, with a reference sequence.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions which are notidentical differ by conservative amino acid substitutions.

The terms “variant” and “mutant” when used in reference to a polypeptiderefer to an amino acid sequence that differs by one or more amino acidsfrom another, usually related polypeptide. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties. One type of conservative amino acidsubstitutions refers to the interchangeability of residues havingsimilar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. More rarely, a variant may have “non-conservative”changes (e.g., replacement of a glycine with a tryptophan). Similarminor variations may also include amino acid deletions or insertions(i.e., additions), or both. Guidance in determining which and how manyamino acid residues may be substituted, inserted or deleted withoutabolishing biological activity may be found using computer programs wellknown in the art, for example, DNAStar software. Variants can be testedin functional assays. Preferred variants have less than 10%, andpreferably less than 5%, and still more preferably less than 2% changes(whether substitutions, deletions, and so on).

The term “domain” when used in reference to a polypeptide refers to asubsection of the polypeptide which possesses a unique structural and/orfunctional characteristic; typically, this characteristic is similaracross diverse polypeptides. The subsection typically comprisescontiguous amino acids, although it may also comprise amino acids whichact in concert or which are in close proximity due to folding or otherconfigurations. An example of a protein domain is the CUB domain inADAMTS13, which has been identified in a number of developmentallyregulated proteins.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises coding sequences necessary for the production of an RNA,or a polypeptide or its precursor (e.g., proinsulin). A functionalpolypeptide can be encoded by a full length coding sequence or by anyportion of the coding sequence as long as the desired activity orfunctional properties (e.g., enzymatic activity, ligand binding, signaltransduction, etc.) of the polypeptide are retained. The term “portion”when used in reference to a gene refers to fragments of that gene. Thefragments may range in size from a few nucleotides to the entire genesequence minus one nucleotide. Thus, “a nucleotide comprising at least aportion of a gene” may comprise fragments of the gene or the entiregene.

The term “gene” also encompasses the coding regions of a structural geneand includes sequences located adjacent to the coding region on both the5′ and 3′ ends for a distance of about 1 kb on either end such that thegene corresponds to the length of the full-length mRNA. The sequenceswhich are located 5′ of the coding region and which are present on themRNA are referred to as 5′ non-translated sequences. The sequences whichare located 3′ or downstream of the coding region and which are presenton the mRNA are referred to as 3′ non-translated sequences. The term“gene” encompasses both cDNA and genomic forms of a gene. A genomic formor clone of a gene contains the coding region interrupted withnon-coding sequences termed “introns” or “intervening regions” or“intervening sequences.” Introns are segments of a gene which aretranscribed into nuclear RNA (hnRNA); introns may contain regulatoryelements such as enhancers. Introns are removed or “spliced out” fromthe nuclear or primary transcript; introns therefore are absent in themessenger RNA (mRNA) transcript. The mRNA functions during translationto specify the sequence or order of amino acids in a nascentpolypeptide.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequenceswhich are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers which control or influence thetranscription of the gene. The 3′ flanking region may contain sequenceswhich direct the termination of transcription, posttranscriptionalcleavage and polyadenylation.

In particular, the term “ADAMTS13 gene” refers to a full-length ADAMTS13nucleotide sequence (e.g., as shown in SEQ ID NO:5). However, it is alsointended that the term encompass fragments of the ADAMTS13 sequence, aswell as other domains with the full-length ADAMTS13 nucleotide sequence.Furthermore, the terms “ADAMTS 13 nucleotide sequence” or “ADAMTS13polynucleotide sequence” encompasses DNA, cDNA, and RNA (e.g., mRNA)sequences.

The term “heterologous” when used in reference to a gene refers to agene encoding a factor that is not in its natural environment (i.e., hasbeen altered by the hand of man). For example, a heterologous geneincludes a gene from one species introduced into another species. Aheterologous gene also includes a gene native to an organism that hasbeen altered in some way (e.g., mutated, added in multiple copies,linked to a non-native promoter or enhancer sequence, etc.).Heterologous genes may comprise plant gene sequences that comprise cDNAforms of a plant gene; the cDNA sequences may be expressed in either asense (to produce mRNA) or anti-sense orientation (to produce ananti-sense RNA transcript that is complementary to the mRNA transcript).Heterologous genes are distinguished from endogenous plant genes in thatthe heterologous gene sequences are typically joined to nucleotidesequences comprising regulatory elements such as promoters that are notfound naturally associated with the gene for the protein encoded by theheterologous gene or with plant gene sequences in the chromosome, or areassociated with portions of the chromosome not found in nature (e.g.,genes expressed in loci where the gene is not normally expressed).

The term “nucleotide sequence of interest” or “nucleic acid sequence ofinterest” refers to any nucleotide sequence (e.g., RNA or DNA), themanipulation of which may be deemed desirable for any reason (e.g.,treat disease, confer improved qualities, etc.), by one of ordinaryskill in the art. Such nucleotide sequences include, but are not limitedto, coding sequences of structural genes (e.g., reporter genes,selection marker genes, oncogenes, drug resistance genes, growthfactors, etc.), and non-coding regulatory sequences which do not encodean mRNA or protein product (e.g., promoter sequence, polyadenylationsequence, termination sequence, enhancer sequence, etc.).

The term “structural” when used in reference to a gene or to anucleotide or nucleic acid sequence refers to a gene or a nucleotide ornucleic acid sequence whose ultimate expression product is a protein(such as an enzyme or a structural protein), an rRNA, an sRNA, a tRNA,etc.

The terms “oligonucleotide” or “polynucleotide” or “nucleotide” or“nucleic acid” refer to a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than three, andusually more than ten. The exact size will depend on many factors, whichin turn depends on the ultimate function or use of the oligonucleotide.The oligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, or a combinationthereof.

The terms “an oligonucleotide having a nucleotide sequence encoding agene” or “a nucleic acid sequence encoding” a specified polypeptiderefer to a nucleic acid sequence comprising the coding region of a geneor in other words the nucleic acid sequence which encodes a geneproduct. The coding region may be present in either a cDNA, genomic DNAor RNA form. When present in a DNA form, the oligonucleotide may besingle-stranded (i.e., the sense strand) or double-stranded. Suitablecontrol elements such as enhancers/promoters, splice junctions,polyadenylation signals, etc. may be placed in close proximity to thecoding region of the gene if needed to permit proper initiation oftranscription and/or correct processing of the primary RNA transcript.Alternatively, the coding region utilized in the expression vectors ofthe present invention may contain endogenous enhancers/promoters, splicejunctions, intervening sequences, polyadenylation signals, etc. or acombination of both endogenous and exogenous control elements.

The term “recombinant” when made in reference to a nucleic acid moleculerefers to a nucleic acid molecule which is comprised of segments ofnucleic acid joined together by means of molecular biologicaltechniques. The term “recombinant” when made in reference to a proteinor a polypeptide refers to a protein molecule which is expressed using arecombinant nucleic acid molecule.

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, for the sequence “A-G-T,” is complementary to the sequence“T-C-A.” Complementarity may be “partial,” in which only some of thenucleic acids' bases are matched according to the base pairing rules.Or, there may be “complete” or “total” complementarity between thenucleic acids. The degree of complementarity between nucleic acidstrands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methodswhich depend upon binding between nucleic acids.

The term “homology” when used in relation to nucleic acids refers to adegree of complementarity. There may be partial homology or completehomology (i.e., identity). “Sequence identity” refers to a measure ofrelatedness between two or more nucleic acids or proteins, and is givenas a percentage with reference to the total comparison length. Theidentity calculation takes into account those nucleotide or amino acidresidues that are identical and in the same relative positions in theirrespective larger sequences. Calculations of identity may be performedby algorithms contained within computer programs such as “GAP” (GeneticsComputer Group, Madison, Wis.) and “ALIGN” (DNAStar, Madison, Wis.). Apartially complementary sequence is one that at least partially inhibits(or competes with) a completely complementary sequence from hybridizingto a target nucleic acid is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a sequence which is completelyhomologous to a target under conditions of low stringency. This is notto say that conditions of low stringency are such that non-specificbinding is permitted; low stringency conditions require that the bindingof two sequences to one another be a specific (i.e., selective)interaction. The absence of non-specific binding may be tested by theuse of a second target which lacks even a partial degree ofcomplementarity (e.g., less than about 30% identity); in the absence ofnon-specific binding the probe will not hybridize to the secondnon-complementary target.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence”, “sequenceidentity”, “percentage of sequence identity”, and “substantialidentity”. A “reference sequence” is a defined sequence used as a basisfor a sequence comparison; a reference sequence may be a subset of alarger sequence, for example, as a segment of a full-length cDNAsequence given in a sequence listing or may comprise a complete genesequence. Generally, a reference sequence is at least 20 nucleotides inlength, frequently at least 25 nucleotides in length, and often at least50 nucleotides in length. Since two polynucleotides may each (1)comprise a sequence (i.e., a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides, and (2) mayfurther comprise a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window”, as usedherein, refers to a conceptual segment of at least 20 contiguousnucleotide positions wherein a polynucleotide sequence may be comparedto a reference sequence of at least 20 contiguous nucleotides andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman (Smith &Waterman (1981) Adv. Appl. Math., 2:482) by the homology alignmentalgorithm of Needleman and Wunsch (Needleman & Wunsch (1970) J. Mol.Biol., 48:443), by the search for similarity method of Pearson andLipman (Pearson & Lipman (1988) Proc. Natl. Acad. Sci. U.S.A., 85:2444),by computerized implementations of these algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package Release7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection, and the best alignment (i.e., resulting in the highestpercentage of homology over the comparison window) generated by thevarious methods is selected. The term “sequence identity” means that twopolynucleotide sequences are identical (i.e., on anucleotide-by-nucleotide basis) over the window of comparison. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. The terms “substantial identity” as used herein denotes acharacteristic of a polynucleotide sequence, wherein the polynucleotidecomprises a sequence that has at least 85 percent sequence identity,preferably at least 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 nucleotide positions, frequentlyover a window of at least 25-50 nucleotides, wherein the percentage ofsequence identity is calculated by comparing the reference sequence tothe polynucleotide sequence which may include deletions or additionswhich total 20 percent or less of the reference sequence over the windowof comparison. The reference sequence may be a subset of a largersequence, for example, as a segment of the full-length sequences of thecompositions claimed in the present invention.

The term “substantially homologous” when used in reference to adouble-stranded nucleic acid sequence such as a cDNA or genomic clonerefers to any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low to highstringency as described above.

The term “substantially homologous” when used in reference to asingle-stranded nucleic acid sequence refers to any probe that canhybridize (i.e., it is the complement of) the single-stranded nucleicacid sequence under conditions of low to high stringency as describedabove.

The term “hybridization” refers to the pairing of complementary nucleicacids. Hybridization and the strength of hybridization (i.e., thestrength of the association between the nucleic acids) is impacted bysuch factors as the degree of complementary between the nucleic acids,stringency of the conditions involved, the T_(m) of the formed hybrid,and the G:C ratio within the nucleic acids. A single molecule thatcontains pairing of complementary nucleic acids within its structure issaid to be “self-hybridized.”

The term “T_(m)” refers to the “melting temperature” of a nucleic acid.The melting temperature is the temperature at which a population ofdouble-stranded nucleic acid molecules becomes half dissociated intosingle strands. The equation for calculating the T_(m) of nucleic acidsis well known in the art. As indicated by standard references, a simpleestimate of the T_(m) value may be calculated by the equation:T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1M NaCl (See e.g., Anderson and Young, Quantitative Filter Hybridization,in Nucleic Acid Hybridization (1985)). Other references include moresophisticated computations that take structural as well as sequencecharacteristics into account for the calculation of T_(m).

The term “stringency” refers to the conditions of temperature, ionicstrength, and the presence of other compounds such as organic solvents,under which nucleic acid hybridizations are conducted. With “highstringency” conditions, nucleic acid base pairing will occur onlybetween nucleic acid fragments that have a high frequency ofcomplementary base sequences. Thus, conditions of “low” stringency areoften required with nucleic acids that are derived from organisms thatare genetically diverse, as the frequency of complementary sequences isusually less.

“Low stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42EC in a solution consisting of 5× SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS,5× Denhardt's reagent (50× Denhardt's contains per 500 ml: 5 g Ficoll(Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)) and 100 μg/mldenatured salmon sperm DNA followed by washing in a solution comprising5× SSPE, 0.1% SDS at 42EC when a probe of about 500 nucleotides inlength is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42EC in a solution consisting of 5× SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0× SSPE, 1.0% SDS at 42EC when aprobe of about 500 nucleotides in length is employed.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42EC in a solution consisting of 5× SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1× SSPE, 1.0% SDS at 42EC when aprobe of about 500 nucleotides in length is employed.

It is well known that numerous equivalent conditions may be employed tocomprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (e.g., thepresence or absence of formamide, dextran sulfate, polyethylene glycol)are considered and the hybridization solution may be varied to generateconditions of low stringency hybridization different from, butequivalent to, the above listed conditions. In addition, the art knowsconditions that promote hybridization under conditions of highstringency (e.g., increasing the temperature of the hybridization and/orwash steps, the use of formamide in the hybridization solution, etc.).

The term “wild-type” when made in reference to a gene refers to a genethat has the characteristics of a gene isolated from a naturallyoccurring source. The term “wild-type” when made in reference to a geneproduct refers to a gene product that has the characteristics of a geneproduct isolated from a naturally occurring source. The term“naturally-occurring” as applied to an object refers to the fact that anobject can be found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism (includingviruses) that can be isolated from a source in nature and which has notbeen intentionally modified by man in the laboratory isnaturally-occurring. A wild-type gene is frequently that gene which ismost frequently observed in a population and is thus arbitrarilydesignated the “normal” or “wild-type” form of the gene. In contrast,the term “modified” or “mutant” when made in reference to a gene or to agene product refers, respectively, to a gene or to a gene product whichdisplays modifications in sequence and/or functional properties (i.e.,altered characteristics) when compared to the wild-type gene or geneproduct. It is noted that naturally-occurring mutants can be isolated;these are identified by the fact that they have altered characteristicswhen compared to the wild-type gene or gene product.

Thus, the terms “variant” and “mutant” when used in reference to anucleotide sequence refer to an nucleic acid sequence that differs byone or more nucleotides from another, usually related nucleotide acidsequence. A “variation” is a difference between two different nucleotidesequences; typically, one sequence is a reference sequence.

The term “polymorphic locus” refers to a genetic locus present in apopulation that shows variation between members of the population (i.e.,the most common allele has a frequency of less than 0.95). Thus,“polymorphism” refers to the existence of a character in two or morevariant forms in a population. A “single nucleotide polymorphism” (orSNP) refers a genetic locus of a single base which may be occupied byone of at least two different nucleotides. In contrast, a “monomorphiclocus” refers to a genetic locus at which little or no variations areseen between members of the population (generally taken to be a locus atwhich the most common allele exceeds a frequency of 0.95 in the genepool of the population).

A “frameshift mutation” refers to a mutation in a nucleotide sequence,usually resulting from insertion or deletion of a single nucleotide (ortwo or four nucleotides) which results in a change in the correctreading frame of a structural DNA sequence encoding a protein. Thealtered reading frame usually results in the translated amino-acidsequence being changed or truncated.

A “splice mutation” refers to any mutation that affects gene expressionby affecting correct RNA splicing. Splicing mutation may be due tomutations at intron-exon boundaries which alter splice sites.

The term “detection assay” refers to an assay for detecting the presenceor absence of a sequence or a variant nucleic acid sequence (e.g.,mutation or polymorphism in a given allele of a particular gene, ase.g., ADAMTS13 gene), or for detecting the presence or absence of aparticular protein (e.g., ADAMTS13) or the structure or activity oreffect of a particular protein (e.g., VWF-cleaving protease activity) orfor detecting the presence or absence of a variant of a particularprotein.

The term “hybridization analysis” refers to detection of variantnucleotide sequences in a hybridization assay. In a hybridization assay,the presence of absence of a given single nucleotide polymorphism (SNP)or mutation is determined based on the ability of a nucleotide sequencefrom the sample to hybridize to a complementary nucleotide molecule(e.g., a oligonucleotide probe). A variety of hybridization assays usinga variety of technologies for hybridization and detection are available.A description of a selection of exemplary assays is provided later inthe specification, and includes direct detection of hybridization,detection of hybridization using “DNA chip” assays, enzymatic detectionof hybridization, and mass spectroscopic assays of hybridization.

The term “antisense” refers to a deoxyribonucleotide sequence whosesequence of deoxyribonucleotide residues is in reverse 5′ to 3′orientation in relation to the sequence of deoxyribonucleotide residuesin a sense strand of a DNA duplex. A “sense strand” of a DNA duplexrefers to a strand in a DNA duplex which is transcribed by a cell in itsnatural state into a “sense mRNA.” Thus an “antisense” sequence is asequence having the same sequence as the non-coding strand in a DNAduplex. The term “antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene by interfering with theprocessing, transport and/or translation of its primary transcript ormRNA. The complementarity of an antisense RNA may be with any part ofthe specific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, introns, or the coding sequence. In addition, asused herein, antisense RNA may contain regions of ribozyme sequencesthat increase the efficacy of antisense RNA to block gene expression.“Ribozyme” refers to a catalytic RNA and includes sequence-specificendoribonucleases. “Antisense inhibition” refers to the production ofantisense RNA transcripts capable of preventing the expression of thetarget protein.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

Template specificity is achieved in most amplification techniques by thechoice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogeneous mixture of nucleic acid. For example, inthe case of Q_ replicase, MDV-1 RNA is the specific template for thereplicase (Kacian et al. (1972) Proc. Natl. Acad. Sci. USA, 69:3038).Other nucleic acid will not be replicated by this amplification enzyme.Similarly, in the case of T7 RNA polymerase, this amplification enzymehas a stringent specificity for its own promoters (Chamberlain et al.(1970) Nature, 228:227). In the case of T4 DNA ligase, the enzyme willnot ligate the two oligonucleotides or polynucleotides, where there is amismatch between the oligonucleotide or polynucleotide substrate and thetemplate at the ligation junction (Wu & Wallace (1989) Genomics 4:560).Finally, Taq and Pfu polymerases, by virtue of their ability to functionat high temperature, are found to display high specificity for thesequences bounded and thus defined by the primers; the high temperatureresults in thermodynamic conditions that favor primer hybridization withthe target sequences and not hybridization with non-target sequences (H.A. Erlich (ed.), PCR Technology, Stockton Press (1989)).

The term “amplifiable nucleic acid” refers to nucleic acids that may beamplified by any amplification method. It is contemplated that“amplifiable nucleic acid” will usually comprise “sample template.”

The term “sample template” refers to nucleic acid originating from asample that is analyzed for the presence of “target” (defined below). Incontrast, “background template” is used in reference to nucleic acidother than sample template that may or may not be present in a sample.Background template is most often inadvertent. It may be the result ofcarryover, or it may be due to the presence of nucleic acid contaminantssought to be purified away from the sample. For example, nucleic acidsfrom organisms other than those to be detected may be present asbackground in a test sample.

The term “primer” refers to an oligonucleotide, whether occurringnaturally as in a purified restriction digest or produced synthetically,which is capable of acting as a point of initiation of synthesis whenplaced under conditions in which synthesis of a primer extension productwhich is complementary to a nucleic acid strand is induced, (i.e., inthe presence of nucleotides and an inducing agent such as DNA polymeraseand at a suitable temperature and pH). The primer is preferably singlestranded for maximum efficiency in amplification, but may alternativelybe double stranded. If double stranded, the primer is first treated toseparate its strands before being used to prepare extension products.Preferably, the primer is an oligodeoxyribonucleotide. The primer mustbe sufficiently long to prime the synthesis of extension products in thepresence of the inducing agent. The exact lengths of the primers willdepend on many factors, including temperature, source of primer and theuse of the method.

The term “probe” refers to an oligonucleotide (i.e., a sequence ofnucleotides), whether occurring naturally as in a purified restrictiondigest or produced synthetically, recombinantly or by PCR amplification,that is capable of hybridizing to another oligonucleotide of interest. Aprobe may be single-stranded or double-stranded. Probes are useful inthe detection, identification and isolation of particular genesequences. It is contemplated that any probe used in the presentinvention will be labeled with any “reporter molecule,” so that isdetectable in any detection system, including, but not limited to enzyme(e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is not intendedthat the present invention be limited to any particular detection systemor label.

The term “target,” when used in reference to the polymerase chainreaction, refers to the region of nucleic acid bounded by the primersused for polymerase chain reaction. Thus, the “target” is sought to besorted out from other nucleic acid sequences. A “segment” is defined asa region of nucleic acid within the target sequence.

The term “polymerase chain reaction” (“PCR”) refers to the method of K.B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, thatdescribe a method for increasing the concentration of a segment of atarget sequence in a mixture of genomic DNA without cloning orpurification. This process for amplifying the target sequence consistsof introducing a large excess of two oligonucleotide primers to the DNAmixture containing the desired target sequence, followed by a precisesequence of thermal cycling in the presence of a DNA polymerase. The twoprimers are complementary to their respective strands of the doublestranded target sequence. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the target molecule. Following annealing, the primers areextended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing, and polymeraseextension can be repeated many times (i.e., denaturation, annealing andextension constitute one “cycle”; there can be numerous “cycles”) toobtain a high concentration of an amplified segment of the desiredtarget sequence. The length of the amplified segment of the desiredtarget sequence is determined by the relative positions of the primerswith respect to each other, and therefore, this length is a controllableparameter. By virtue of the repeating aspect of the process, the methodis referred to as the “polymerase chain reaction” (hereinafter “PCR”).Because the desired amplified segments of the target sequence become thepredominant sequences (in terms of concentration) in the mixture, theyare said to be “PCR amplified.”

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCTPor dATP, into the amplified segment). In addition to genomic DNA, anyoligonucleotide or polynucleotide sequence can be amplified with theappropriate set of primer molecules. In particular, the amplifiedsegments created by the PCR process itself are, themselves, efficienttemplates for subsequent PCR amplifications.

The terms “PCR product,” “PCR fragment,” and “amplification product”refer to the resultant mixture of compounds after two or more cycles ofthe PCR steps of denaturation, annealing and extension are complete.These terms encompass the case where there has been amplification of oneor more segments of one or more target sequences.

The term “amplification reagents” refers to those reagents(deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template, and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

The term “reverse-transcriptase” or “RT-PCR” refers to a type of PCRwhere the starting material is mRNA. The starting mRNA is enzymaticallyconverted to complementary DNA or “cDNA” using a reverse transcriptaseenzyme. The cDNA is then used as a “template” for a “PCR” reaction

The term “gene expression” refers to the process of converting geneticinformation encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, orsnRNA) through “transcription” of the gene (i.e., via the enzymaticaction of an RNA polymerase), and into protein, through “translation” ofmRNA. Gene expression can be regulated at many stages in the process.“Up-regulation” or “activation” refers to regulation that increases theproduction of gene expression products (i.e., RNA or protein), while“down-regulation” or “repression” refers to regulation that decreaseproduction. Molecules (e.g., transcription factors) that are involved inup-regulation or down-regulation are often called “activators” and“repressors,” respectively.

The terms “in operable combination”, “in operable order” and “operablylinked” refer to the linkage of nucleic acid sequences in such a mannerthat a nucleic acid molecule capable of directing the transcription of agiven gene and/or the synthesis of a desired protein molecule isproduced. The term also refers to the linkage of amino acid sequences insuch a manner so that a functional protein is produced.

The term “regulatory element” refers to a genetic element which controlssome aspect of the expression of nucleic acid sequences. For example, apromoter is a regulatory element which facilitates the initiation oftranscription of an operably linked coding region. Other regulatoryelements are splicing signals, polyadenylation signals, terminationsignals, etc.

Transcriptional control signals in eukaryotes comprise “promoter” and“enhancer” elements. Promoters and enhancers consist of short arrays ofDNA sequences that interact specifically with cellular proteins involvedin transcription (Maniatis, et al. (1987) Science 236:1237). Promoterand enhancer elements have been isolated from a variety of eukaryoticsources including genes in yeast, insect, mammalian and plant cells.Promoter and enhancer elements have also been isolated from viruses andanalogous control elements, such as promoters, are also found inprokaryotes. The selection of a particular promoter and enhancer dependson the cell type used to express the protein of interest. Someeukaryotic promoters and enhancers have a broad host range while othersare functional in a limited subset of cell types (for review, see Voss,et al, Trends Biochem. Sci., 11:287, 1986; and Maniatis, et al., supra1987).

The terms “promoter element,” “promoter,” or “promoter sequence” referto a DNA sequence that is located at the 5′ end (i.e. precedes) of thecoding region of a DNA polymer. The location of most promoters known innature precedes the transcribed region. The promoter functions as aswitch, activating the expression of a gene. If the gene is activated,it is said to be transcribed, or participating in transcription.Transcription involves the synthesis of mRNA from the gene. Thepromoter, therefore, serves as a transcriptional regulatory element andalso provides a site for initiation of transcription of the gene intomRNA.

The term “regulatory region” refers to a gene's 5′ transcribed butuntranslated regions, located immediately downstream from the promoterand ending just prior to the translational start of the gene.

The term “promoter region” refers to the region immediately upstream ofthe coding region of a DNA polymer, and is typically between about 500bp and 4 kb in length, and is preferably about 1 to 1.5 kb in length.

Promoters may be tissue specific or cell specific. The term “tissuespecific” as it applies to a promoter refers to a promoter that iscapable of directing selective expression of a nucleotide sequence ofinterest to a specific type of tissue (e.g., seeds) in the relativeabsence of expression of the same nucleotide sequence of interest in adifferent type of tissue (e.g., leaves). Tissue specificity of apromoter may be evaluated by, for example, operably linking a reportergene to the promoter sequence to generate a reporter construct,introducing the reporter construct into the genome of a plant such thatthe reporter construct is integrated into every tissue of the resultingtransgenic plant, and detecting the expression of the reporter gene(e.g., detecting mRNA, protein, or the activity of a protein encoded bythe reporter gene) in different tissues of the transgenic plant. Thedetection of a greater level of expression of the reporter gene in oneor more tissues relative to the level of expression of the reporter genein other tissues shows that the promoter is specific for the tissues inwhich greater levels of expression are detected. The term “cell typespecific” as applied to a promoter refers to a promoter which is capableof directing selective expression of a nucleotide sequence of interestin a specific type of cell in the relative absence of expression of thesame nucleotide sequence of interest in a different type of cell withinthe same tissue. The term “cell type specific” when applied to apromoter also means a promoter capable of promoting selective expressionof a nucleotide sequence of interest in a region within a single tissue.Cell type specificity of a promoter may be assessed using methods wellknown in the art, e.g., immunohistochemical staining. Briefly, tissuesections are embedded in paraffin, and paraffin sections are reactedwith a primary antibody which is specific for the polypeptide productencoded by the nucleotide sequence of interest whose expression iscontrolled by the promoter. A labeled (e.g., peroxidase conjugated)secondary antibody which is specific for the primary antibody is allowedto bind to the sectioned tissue and specific binding detected (e.g.,with avidin/biotin) by microscopy.

Promoters may be constitutive or inducible. The term “constitutive” whenmade in reference to a promoter means that the promoter is capable ofdirecting transcription of an operably linked nucleic acid sequence inthe absence of a stimulus (e.g., heat shock, chemicals, light, etc.).Typically, constitutive promoters are capable of directing expression ofa transgene in substantially any cell and any tissue. Exemplaryconstitutive plant promoters include, but are not limited to SDCauliflower Mosaic Virus (CaMV SD; see e.g., U.S. Pat. No. 5,352,605,incorporated herein by reference), mannopine synthase, octopine synthase(ocs), superpromoter (see e.g., WO 95/14098), and ubi3 (see e.g.,Garbarino and Belknap, Plant Mol. Biol. 24:119-127 (1994)) promoters.Such promoters have been used successfully to direct the expression ofheterologous nucleic acid sequences in transformed plant tissue.

In contrast, an “inducible” promoter is one which is capable ofdirecting a level of transcription of an operably linked nucleic acidsequence in the presence of a stimulus (e.g., heat shock, chemicals,light, etc.) which is different from the level of transcription of theoperably linked nucleic acid sequence in the absence of the stimulus.

The term “regulatory element” refers to a genetic element that controlssome aspect of the expression of nucleic acid sequence(s). For example,a promoter is a regulatory element that facilitates the initiation oftranscription of an operably linked coding region. Other regulatoryelements are splicing signals, polyadenylation signals, terminationsignals, etc.

The enhancer and/or promoter may be “endogenous” or “exogenous” or“heterologous.” An “endogenous” enhancer or promoter is one that isnaturally linked with a given gene in the genome. An “exogenous” or“heterologous” enhancer or promoter is one that is placed injuxtaposition to a gene by means of genetic manipulation (i.e.,molecular biological techniques) such that transcription of the gene isdirected by the linked enhancer or promoter. For example, an endogenouspromoter in operable combination with a first gene can be isolated,removed, and placed in operable combination with a second gene, therebymaking it a “heterologous promoter” in operable combination with thesecond gene. A variety of such combinations are contemplated (e.g., thefirst and second genes can be from the same species, or from differentspecies).

The term “naturally linked” or “naturally located” when used inreference to the relative positions of nucleic acid sequences means thatthe nucleic acid sequences exist in nature in the relative positions.

The presence of “splicing signals” on an expression vector often resultsin higher levels of expression of the recombinant transcript ineukaryotic host cells. Splicing signals mediate the removal of intronsfrom the primary RNA transcript and consist of a splice donor andacceptor site (Sambrook, et al., Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor Laboratory Press, New York (1989) pp.16.7-16.8). A commonly used splice donor and acceptor site is the splicejunction from the 16S RNA of SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires expression of signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length. The term “poly(A) site” or“poly(A) sequence” as used herein denotes a DNA sequence which directsboth the termination and polyadenylation of the nascent RNA transcript.Efficient polyadenylation of the recombinant transcript is desirable, astranscripts lacking a poly(A) tail are unstable and are rapidlydegraded. The poly(A) signal utilized in an expression vector may be“heterologous” or “endogenous.” An endogenous poly(A) signal is one thatis found naturally at the 3′ end of the coding region of a given gene inthe genome. A heterologous poly(A) signal is one which has been isolatedfrom one gene and positioned 3′ to another gene. A commonly usedheterologous poly(A) signal is the SV40 poly(A) signal. The SV40 poly(A)signal is contained on a 237 bp BamHI/BclI restriction fragment anddirects both termination and polyadenylation (Sambrook, supra, at16.6-16.7).

The term “vector” refers to nucleic acid molecules that transfer DNAsegment(s) from one cell to another. The term “vehicle” is sometimesused interchangeably with “vector.”

The terms “expression vector” or “expression cassette” refer to arecombinant DNA molecule containing a desired coding sequence andappropriate nucleic acid sequences necessary for the expression of theoperably linked coding sequence in a particular host organism. Nucleicacid sequences necessary for expression in prokaryotes usually include apromoter, an operator (optional), and a ribosome binding site, oftenalong with other sequences. Eukaryotic cells are known to utilizepromoters, enhancers, and termination and polyadenylation signals.

The term “transfection” refers to the introduction of foreign DNA intocells. Transfection may be accomplished by a variety of means known tothe art including calcium phosphate-DNA co-precipitation,DEAE-dextran-mediated transfection, polybrene-mediated transfection,glass beads, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, viral infection, biolistics (i.e.,particle bombardment) and the like.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transfected cell. The foreign DNApersists in the nucleus of the transfected cell for several days. Duringthis time the foreign DNA is subject to the regulatory controls thatgovern the expression of endogenous genes in the chromosomes. The term“transient transfectant” refers to cells that have taken up foreign DNAbut have failed to integrate this DNA.

The term “calcium phosphate co-precipitation” refers to a technique forthe introduction of nucleic acids into a cell. The uptake of nucleicacids by cells is enhanced when the nucleic acid is presented as acalcium phosphate-nucleic acid co-precipitate. The original technique ofGraham and van der Eb (Graham & van der Eb (1973) Virol., 52:456), hasbeen modified by several groups to optimize conditions for particulartypes of cells. The art is well aware of these numerous modifications.

The terms “infecting” and “infection” when used with a bacterium referto co-incubation of a target biological sample, (e.g., cell, tissue,etc.) with the bacterium under conditions such that nucleic acidsequences contained within the bacterium are introduced into one or morecells of the target biological sample.

The terms “bombarding, “bombardment,” and “biolistic bombardment” referto the process of accelerating particles towards a target biologicalsample (e.g., cell, tissue, etc.) to effect wounding of the cellmembrane of a cell in the target biological sample and/or entry of theparticles into the target biological sample. Methods for biolisticbombardment are known in the art (e.g., U.S. Pat. No. 5,584,807, thecontents of which are incorporated herein by reference), and arecommercially available (e.g., the helium gas-driven microprojectileaccelerator (PDS-1000/He, BioRad).

The term “transgene” refers to a foreign gene that is placed into anorganism by the process of transfection. The term “foreign gene” refersto any nucleic acid (e.g., gene sequence) that is introduced into thegenome of an organism by experimental manipulations and may include genesequences found in that organism so long as the introduced gene does notreside in the same location as does the naturally-occurring gene.

The term “transgenic” when used in reference to a host cell or anorganism refers to a host cell or an organism that contains at least oneheterologous or foreign gene in the host cell or in one or more of cellsof the organism.

The term “host cell” refers to any cell capable of replicating and/ortranscribing and/or translating a heterologous gene. Thus, a “host cell”refers to any eukaryotic or prokaryotic cell (e.g., bacterial cells suchas E. coli, yeast cells, mammalian cells, avian cells, amphibian cells,plant cells, fish cells, and insect cells), whether located in vitro orin vivo. For example, host cells may be located in a transgenic animal.

The terms “transformants” or “transformed cells” include the primarytransformed cell and cultures derived from that cell without regard tothe number of transfers. All progeny may not be precisely identical inDNA content, due to deliberate or inadvertent mutations. Mutant progenythat have the same functionality as screened for in the originallytransformed cell are included in the definition of transformants.

The term “selectable marker” refers to a gene which encodes an enzymehaving an activity that confers resistance to an antibiotic or drug uponthe cell in which the selectable marker is expressed, or which confersexpression of a trait which can be detected (e.g., luminescence orfluorescence). Selectable markers may be “positive” or “negative.”Examples of positive selectable markers include the neomycinphosphotrasferase (NPTII) gene which confers resistance to G418 and tokanamycin, and the bacterial hygromycin phosphotransferase gene (hyg),which confers resistance to the antibiotic hygromycin. Negativeselectable markers encode an enzymatic activity whose expression iscytotoxic to the cell when grown in an appropriate selective medium. Forexample, the HSV-tk gene is commonly used as a negative selectablemarker. Expression of the HSV-tk gene in cells grown in the presence ofgancyclovir or acyclovir is cytotoxic; thus, growth of cells inselective medium containing gancyclovir or acyclovir selects againstcells capable of expressing a functional HSV TK enzyme.

The term “reporter gene” refers to a gene encoding a protein that may beassayed. Examples of reporter genes include, but are not limited to,luciferase (See, e.g., deWet et al., Mol. Cell. Biol. 7:725 (1987) andU.S. Pat. Nos., 6,074,859; 5,976,796; 5,674,713; and 5,618,682; all ofwhich are incorporated herein by reference), green fluorescent protein(e.g., GenBank Accession Number U43284; a number of GFP variants arecommercially available from CLONTECH Laboratories, Palo Alto, Calif.),chloramphenicol acetyltransferase, β-galactosidase, alkalinephosphatase, and horse radish peroxidase.

The term “overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. The term “cosuppression” refers to theexpression of a foreign gene which has substantial homology to anendogenous gene resulting in the suppression of expression of both theforeign and the endogenous gene. As used herein, the term “alteredlevels” refers to the production of gene product(s) in transgenicorganisms in amounts or proportions that differ from that of normal ornon-transformed organisms.

The terms “Southern blot analysis” and “Southern blot” and “Southern”refer to the analysis of DNA on agarose or acrylamide gels in which DNAis separated or fragmented according to size followed by transfer of theDNA from the gel to a solid support, such as nitrocellulose or a nylonmembrane. The immobilized DNA is then exposed to a labeled probe todetect DNA species complementary to the probe used. The DNA may becleaved with restriction enzymes prior to electrophoresis. Followingelectrophoresis, the DNA may be partially depurinated and denaturedprior to or during transfer to the solid support. Southern blots are astandard tool of molecular biologists (J. Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, pp9.31-9.58).

The term “Northern blot analysis” and “Northern blot” and “Northern”refer to the analysis of RNA by electrophoresis of RNA on agarose gelsto fractionate the RNA according to size followed by transfer of the RNAfrom the gel to a solid support, such as nitrocellulose or a nylonmembrane. The immobilized RNA is then probed with a labeled probe todetect RNA species complementary to the probe used. Northern blots are astandard tool of molecular biologists (J. Sambrook, et al. (1989) supra,pp 7.39-7.52).

The terms “Western blot analysis” and “Western blot” and “Western”refers to the analysis of protein(s) (or polypeptides) immobilized ontoa support such as nitrocellulose or a membrane. A mixture comprising atleast one protein is first separated on an acrylamide gel, and theseparated proteins are then transferred from the gel to a solid support,such as nitrocellulose or a nylon membrane. The immobilized proteins areexposed to at least one antibody with reactivity against at least oneantigen of interest. The bound antibodies may be detected by variousmethods, including the use of radiolabeled antibodies.

The term “antigenic determinant” refers to that portion of an antigenthat makes contact with a particular antibody (i.e., an epitope). When aprotein or fragment of a protein is used to immunize a host animal,numerous regions of the protein may induce the production of antibodiesthat bind specifically to a given region or three-dimensional structureon the protein; these regions or structures are referred to as antigenicdeterminants. An antigenic determinant may compete with the intactantigen (i.e., the “immunogen” used to elicit the immune response) forbinding to an antibody.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” refers to a nucleic acid sequence that isidentified and separated from at least one contaminant nucleic acid withwhich it is ordinarily associated in its natural source. Isolatednucleic acid is present in a form or setting that is different from thatin which it is found in nature. In contrast, non-isolated nucleic acids,such as DNA and RNA, are found in the state they exist in nature.Examples of non-isolated nucleic acids include: a given DNA sequence(e.g., a gene) found on the host cell chromosome in proximity toneighboring genes; RNA sequences, such as a specific mRNA sequenceencoding a specific protein, found in the cell as a mixture withnumerous other mRNAs which encode a multitude of proteins. However,isolated nucleic acid encoding a particular protein includes, by way ofexample, such nucleic acid in cells ordinarily expressing the protein,where the nucleic acid is in a chromosomal location different from thatof natural cells, or is otherwise flanked by a different nucleic acidsequence than that found in nature. The isolated nucleic acid oroligonucleotide may be present in single-stranded or double-strandedform. When an isolated nucleic acid or oligonucleotide is to be utilizedto express a protein, the oligonucleotide will contain at a minimum thesense or coding strand (i.e., the oligonucleotide may single-stranded),but may contain both the sense and anti-sense strands (i.e., theoligonucleotide may be double-stranded).

The term “purified” refers to molecules, either nucleic or amino acidsequences, that are removed from their natural environment, isolated orseparated. An “isolated nucleic acid sequence” may therefore be apurified nucleic acid sequence. “Substantially purified” molecules areat least 60% free, preferably at least 75% free, and more preferably atleast 90% free from other components with which they are naturallyassociated. As used herein, the term “purified” or “to purify” alsorefer to the removal of contaminants from a sample. The removal ofcontaminating proteins results in an increase in the percent ofpolypeptide of interest in the sample. In another example, recombinantpolypeptides are expressed in plant, bacterial, yeast, or mammalian hostcells and the polypeptides are purified by the removal of host cellproteins; the percent of recombinant polypeptides is thereby increasedin the sample.

The term “composition comprising” a given polynucleotide sequence orpolypeptide refers broadly to any composition containing the givenpolynucleotide sequence or polypeptide. The composition may comprise anaqueous solution. Compositions comprising polynucleotide sequencesencoding ADAMTS13 (e.g., SEQ ID NO:2) or fragments thereof may beemployed as hybridization probes. In this case, the ADAMTS13 encodingpolynucleotide sequences are typically employed in an aqueous solutioncontaining salts (e.g., NaCl), detergents (e.g., SDS), and othercomponents (e.g., Denhardt's solution, dry milk, salmon sperm DNA,etc.).

The term “test compound” refers to any chemical entity, pharmaceutical,drug, and the like that can be used to treat or prevent a disease,illness, sickness, or disorder of bodily function, or otherwise alterthe physiological or cellular status of a sample. Test compoundscomprise both known and potential therapeutic compounds. A test compoundcan be determined to be therapeutic by screening using the screeningmethods of the present invention. A “known therapeutic compound” refersto a therapeutic compound that has been shown (e.g., through animaltrials or prior experience with administration to humans) to beeffective in such treatment or prevention.

As used herein, the term “response,” when used in reference to an assay,refers to the generation of a detectable signal (e.g., accumulation ofreporter protein, increase in ion concentration, accumulation of adetectable chemical product).

The term “sample” is used in its broadest sense. In one sense it canrefer to a plant cell or tissue. In another sense, it is meant toinclude a specimen or culture obtained from any source, as well asbiological and environmental samples. Biological samples may be obtainedfrom plants or animals (including humans) and encompass fluids, solids,tissues, and gases. Environmental samples include environmental materialsuch as surface matter, soil, water, and industrial samples. Theseexamples are not to be construed as limiting the sample types applicableto the present invention.

GENERAL DESCRIPTION OF THE INVENTION

Although the cause of TTP is unknown, some evidence suggested that thetreatment resulted from removal of a toxic factor from the blood, whileother evidence suggested that it replaced a missing factor. Recentevidence suggested that the missing factor could be a type of proteincalled a protease, and in particular a protease which degrades anotherblood clotting factor called von Willebrand factor (VWF). In 1982, Moakeet al (Moake et al. (1982) N. Engl. J. Med. 307, 1432-1435) observedunusually large multimeric forms of von Willebrand factor (VWF) in theplasma of TTP patients and postulated that these patients may lack anactivity that is responsible for decreasing the size of VWF secretedfrom endothelial cells. In 1996, two groups independently isolated aprotease from plasma that appears to be responsible for the physiologiccleavage of VWF at the Tyr842-Met843 peptide bond, producing thecharacteristic 176 kd and 140 kd proteolytic fragments observed innormal plasma (Tsai, H. M. (1996) Blood 87, 4235-4244; Furlan et al.(1996) Blood 87, 4223-4234). Increased susceptibility to thisproteolytic cleavage appears to be responsible for the loss of large VWFmultimers central to the pathophysiology of a different disease, type 2Avon Willebrand disease (VWD) (Tsai et al. (1997) Blood 89, 1954-1962).The same protease activity was subsequently shown to be deficient in theplasma of TTP patients (Tsai and Lian (1998) N. Engl. J. Med. 339,1585-1594; Furlan et al. (1998) N. Engl. J. Med. 339, 1578-1584). Thehypothesis that the disease results from the presence of a toxic factorin the blood is supported by reports of circulating autoantibodiesdetected in most adults with disease (Tsai and Lian (1998) N. Engl. J.Med. 339, 1585-1594; Furlan et al. (1998) N. Engl. J. Med. 339,1578-1584), as well as recent reports of antibodies against thisprotease which been identified in a form of TTP associated with theantiplatelet drug ticlopidine(Tsai et al. (2000) Ann. Intern. Med. 132,794-799).

Despite the strong association of low VWF-cleaving protease activitywith TTP, a direct causative link has not yet been established. Otherstudies have implicated platelet aggregating proteins or endothelialinjury as the underlying mechanism (Mitra et al. (1997) Blood 89,1224-1234); Dang et al. (1999) Blood 93, 1264-1270; Cines et al. (2000)Thromb. Haemost. 84, 528-535) and enhanced rather than decreased VWFproteolysis has been observed in some patients (Mannucci et al. (1989)Blood 74, 978-983). Though the protease responsible for VWF cleavage hasbeen partially purified and characterized (Tsai et al. (1997) Blood 89,1954-1962; Furlan et al (1996) Blood 87, 4223-4234), it appears to bepresent at relatively low levels in plasma and its identification at thesequence level has remained elusive.

The present invention provides identification and characterization ofthe gene responsible for familial TTP. This was accomplished by studyinga series of families in which TTP appears to be inherited and then usinga positional cloning approach to map a gene responsible for reducedVWF-cleaving protease activity to a locus on 9q34. The gene wasidentified as ADAMTS13 which encodes ADAMTS13, a unique member of themetalloproteinase gene family. Expression of ADAMTS13 from clonedfull-length cDNA confirmed its VWF-cleaving protease activity. At leasttwo different forms of ADAMTS13 have been identified, which vary inlength. Moreover, mutations in this gene were discovered in individualsaffected with TTP. All but 3 of 13 ADAMTS13 mutations identified weremissense mutations. Moreover, the two frameshift and one splicemutations identified were present in trans with a missense mutation onthe other allele, which suggests that complete deficiency of ADAMTS13may be lethal. Nine TTP-related ADAMTS13 missense mutations severelyimpair VWF-cleaving protease activity, accounting for the loss ofactivity observed in the corresponding patient plasmas.

Thus, the present invention provides nucleotide sequences encodingwild-type, mutants, variants, and fragments of ADAMTS13, as well as theencoded proteins. The present invention further provides methods ofusing the ADAMTS13 gene and protein, which include but are not limitedto precise and rapid diagnosis of this condition in other individualswith inherited TTP, such as with nucleic acid probes or with antibodies,treatment of patients with TTP with a recombinant ADAMTS13, andtreatment of patients at risk of or suffering from heart attack orstroke with this protease or other drugs developed from this proteasewhich act as anticoagulants.

In the following description of the discovery and characterization ofthe ADAMTS13 gene, mutants, and variants, hypotheses may be advanced toexplain certain results, or to correlate results with previousobservations. It is not necessary to understand the mechanism underlyingthe invention, nor is it intended that the invention be limited to anyparticular mechanism.

A. Discovery of the ADAMTS13 Gene

1. Analysis of Plasma Level of VWF-Cleaving Protease.

Four pedigrees of families in which TTP appears to be inherited wereavailable for analysis, and are shown in FIG. 1. The levels of plasmaVWF-cleaving protease were analyzed as described in Example 1B; theresults indicated that the plasma level of VWF-cleaving proteasesegregated as a semidominant autosomal trait.

VWF-cleaving protease activity measured in the plasma of the 7 affectedindividuals ranged from 2-7% of normal (0.02-0.07 U/ml) and none of thepatients tested positive for inhibitors of the protease. Plasma proteaselevels in the parents of the affected individuals ranged from 0.51-0.68U/ml, consistent with a heterozygous carrier state. Similarly, levelsfor at-risk siblings of the patients and parents fell into a bimodaldistribution, with one peak consistent with carriers and the otherindistinguishable from the normal distribution (FIG. 2). These resultsdemonstrate that the protease activity assay used here reliablydistinguishes between normal and carrier individuals in these families.This observation suggested that the plasma level of VWF-cleavingprotease could be used as a phenotypic trait for linkage analysis to mapthe corresponding locus, providing considerably greater genetic powerthan would be available from analysis of the clinical phenotype alone.

2. Mapping the Gene for Familial TTP to Chromosome 9q34.

A genome wide linkage scan was thus performed on the four pedigreesshown in FIG. 1 using 382 polymorphic microsatellite markers to analyzeDNA from affected individuals and other informative family members, asdescribed in Example 1. Two-point linkage analysis using a recessivemodel gave a maximum LOD score of 2.36 at θ=0.0 for marker D9S164 onchromosome 9q34, with a LOD score of 3.83 at θ=0.01 for a codominantmodel. Multipoint analysis for D9S164 and 4 flanking markers(cen-D9S1682-D9S290-D95164-D9S1826-D9S158-tel) yielded a maximum LODscore of 4.77 at a location 2.4 cM telomeric to marker D9S164. Genotypesfor 7 other markers in this region (Dib, C. et al. (1996) Nature 380,152-154;. Broman, K. W. et al. (1998) Am. J. Hum. Genet. 63, 861-869)allowed the gene to be placed in the ˜7 cM interval between markersD9S1863 and D9S1818 (FIGS. 1 and 3A). Analysis of additional polymorphicmarkers (see Table 3 in Example 1) designed from simple sequence repeatdata available from the Human Genome Working Draft narrowed thecandidate interval to an ˜2.3 Mb genomic segment between markers GL2-1and D9S1818. In all but one case, carrier status as determined byhaplotype analysis was consistent with the phenotypic designationaccording to plasma protease level. The exception, individual II2 inpedigree A, shares the affected haplotype of her brother (II4), but hasa protease level of 0.8 U/ml, which is borderline between the normal andcarrier ranges.

3. Identification of a Candidate Gene for Familial TTP.

Analysis of the candidate interval using public genome databaseresources identified ˜20 known or predicted genes (FIG. 3A). Initialattention focused on genes likely to encode a protease or proteasecofactor. FCN2 (ficolin 2) mapped to distal chromosome 9 but could notbe identified in available BAC sequence from the candidate interval.However, in light of previous reports suggesting a protease associatedfunction for some ficolin family members (Matsushita, M. & Fujita, T.Ficolins (2001) Immunol. Rev. 180, 78-85) and the possibility that FCN2might lie in one of the three large genomic sequence gaps shown in FIG.3A, the coding exons and intron/exon boundaries of this gene wereamplified by PCR from patient DNA and subjected to sequence analysis. Nocandidate mutations were identified. Two putative genes in the candidateinterval, KIAA0605, an uncharacterized EST from a brain cDNA library(Nagase, T. et al. (1998) DNA Res. 5, 31-39), and the predicted openreading frame C9ORF8, exhibited homology to the ADAMTS family ofmetalloproteinases, but appeared to lack the conserved proteasecatalytic domain. Partial DNA sequence analysis of exons and flankingintron sequences failed to identify any mutations in KIAA0605. However,the identification of several candidate missense mutations in thepredicted exons of C9ORF8 led to further, more detailed analysis of thiscandidate gene.

Exon 1 of C9ORF8 overlapped with a cluster of EST sequences (Unigenecluster Hs.149184), predicting a large 5′ untranslated region. A segmentof putative C9ORF8 coding sequence was used to probe a human fetal cDNAlibrary identifying several partial cDNA clones, which were extended inboth the 5′ and 3′ direction by RT-PCR and RACE. The assembled cDNAsequence corrected an error in the predicted boundaries of C9ORF8 exon2, resulting in a continuous open reading frame including two exonsupstream of the 5′ EST cluster, 3 new exons within the predicted intron10 of C9ORF8 and 6 additional downstream exons overlapping a secondhypothetical gene in this region, DKFZp434C2322 (Unigene clusterHs.131433). Thus, through a combination of cDNA cloning, RACE, andgenomic sequence analysis, the full length cDNA sequence (FIG. 5) andcorresponding genomic structure were deduced, as depicted in FIG. 3B,and found to encode a complete, potentially catalytically active ADAMTSprotease (FIG. 6). This gene was discovered to be a novel member of theADAMTS family of metalloproteases, and was therefore designatedADAMTS13.

B. Characterization of ADAMTS13 Gene and ADAMTS13 Protein

ADAM (A Disintegrin And Metalloproteinase) family members aremembrane-anchored proteases with diverse functions. Known membersinclude fertilins α and β, implicated in sperm-egg fusion, and the“sheddases” such as TACE (TNFα convertase), which mediate the sheddingof cell surface proteins (Blobel, C. P. (1997) Cell 90, 589-592). ADAMTSfamily members are distinguished from ADAMs by the presence of one ormore thrombospondin 1-like (TSP1) domain(s) at the C-terminus and by theabsence of the EGF repeat, transmembrane domain and cytoplasmic tailtypically observed in ADAM metalloproteinases. The TSP1 motifs arethought to mediate interactions with components of the extracellularmatrix (Kaushal, G. P. & Shah, S. V. (2000) J. Clin. Invest 105,1335-1337; Hurskainen, T. L. et al. (1999) J. Biol. Chem. 274,25555-25563; and Tang, B. L. (2001) Int. J. Biochem. Cell Biol. 33,33-44). ADAMTS4 and 5/11 (aggrecanases) cleave the proteoglycan core ofarticular cartilage and may play a role in inflammatory joint disease(Tortorella, M. D. et al (1999) Science 284, 1664-1666). and mutationsin ADAMTS2 (procollagen N-proteinase) result in the connective tissuedisorder Ehlers-Danlos Syndrome, Type V (Colige, A. et al. (1999) Am. J.Hum. Genet. 65, 308-317). Though ADAMTS1 mutations have not beenidentified in humans, genetically deficient mice exhibit growthretardation, adipose tissue abnormalities, and fibrotic changesthroughout the genitourinary system, suggesting a critical role forADAMTS1 in organogenesis and tissue remodeling (Shindo, T. et al. (2000)J. Clin. Invest. 105, 1345-1352). The function and protein substratesfor the remaining ADAMTS family members are unknown.

1. ADAMTS13 Coding Sequence.

The full-length ADAMTS13 mRNA is 4,550 nucleotides in length, encoding a1,427 amino acid open reading frame that begins with the first ATG,leaving short 5′ and 3′ untranslated regions of 61 bp and 208 bp,respectively. The ADAMTS13 gene spans 29 exons encompassingapproximately 37 kb in the human genome and encoding a 1,427 amino acidprotein (FIG. 3B). Analysis of RT-PCR and cloned cDNA sequences providedevidence for alternative splicing of exon 17, resulting in a frameshiftthat predicts a truncated 842 amino acid form of the protein lacking the6 C-terminal TSP1 repeats (as shown in FIGS. 7 and 8). Comparativeanalysis with draft mouse genomic sequences demonstrates a high degreeof conservation throughout the coding exons and identifies an additionalpotential exon located between the current exons 22 and 23, which mayindicate another splice isoform. These findings suggest the potentialfor differentially regulated alternative isoforms of ADAMTS13 withdiverse biologic functions in addition to the proteolytic processing ofVWF. Alternative splicing has also been observed in other ADAMTSproteins, including ADAMTS9, resulting in a similar variation in thenumber of C-terminal TSP1 repeats (Tang, B. L. (2001) Int. J. Biochem.Cell Biol. 33, 33-44).

2. ADAMTS13 protein

The domain structure of ADAMTS13 is depicted at the bottom of FIG. 3B. Apredicted signal peptide is followed by a short propeptide domain endingin a potential propeptide convertase cleavage site at amino acids 71-74(RQRR), suggesting that proteolytic processing, either in the transGolgi or at the cell surface, is required for activation. The proteasedomain that follows contains a perfect match for the HEXGHXXGXXHDextended catalytic site consensus sequence shared between snake venommetalloproteinases, and ADAM family members (Kaushal, G. P. & Shah, S.V. (2000) J. Clin. Invest 105: 1335-1337; Blobel, C. P. (1997) Cell 90,589-592; and Kuno, K. et al. (1997). J. Biol. Chem. 272, 556-562). Thecatalytic domain is followed by the disintegrin, thrombospondin type 1(TSP1), and spacer domains characteristic of the ADAMTS family. An RGDSsequence not present in other ADAMTSs is located immediately C-terminalto the first TSP1 domain, suggesting a possible novel integrininteraction. The C-terminus contains an additional 6 TSP1 repeats,followed by a segment with homology to a CUB domain. CUB domains havebeen identified in a number of developmentally regulated proteins (Bork,P. & Beckmann, G. (1993) J. Mol. Biol. 231, 539-545); however, thisdomain has not been reported for an ADAMTS protein, and appears to benovel to ADAMTS13. The previously reported inhibitor profile and metalcation dependence of the VWF-cleaving protease (Tsai, H. M. (1996) Blood87, 4235-4244; Furlan, et al. (1996) Blood 87, 4223-4234; Tsai, et al.(1997) Blood 89, 1954-1962) are consistent with its identity as anADAMTS. The predicted, nonglycosylated molecular mass of ADAMTS13 is 154kd, consistent with a previously estimated mass of 200 kd for partiallypurified VWF-cleaving protease (Tsai, H. M. (1996) Blood 87, 4235-4244),though considerably smaller than the 300 kd mass reported by other(Furlan et al. (1996) Blood 87, 4223-4234).

3. ADAMTS13 Expression and Activity

The full-length ADAMTS13 cDNA was assembled and cloned into a mammalianexpression vector and transfected into CHO-Tag cells (as described inthe Examples). Conditioned medium from transfected cells was tested forVWF-cleaving protease activity by a previously-described assay (Tsai etal. (2001) Clin. Lab 47, 387-392) and was found to exhibit aVWF-cleaving protease activity of 0.47 U (±0.07), as compared to a valueof 0.06 (±0.03) in conditioned media from mock-transfected cells(p<0.01). These data directly demonstrate the VWF-cleaving proteaseactivity of recombinant ADAMTS13.

These results demonstrate the feasibility of producing recombinantADAMTS13 and confirm that the latter possesses VWF-cleaving proteaseactivity. The VWF-cleaving protease assay used here (Tsai et al. (2001)Clin. Lab 47, 387-392) relies on the detection of the 176 kD dimerformed by VWF cleavage at the peptide bond between Tyr842 and Met843(Dent et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6306-6310),further indicating that cleavage of VWF by recombinant ADAMTS13 occursat or near this bond. These results support the use of providing anactive form ADAMTS13 for the treatment of TTP; moreover, it iscontemplated that the production of recombinant protein will facilitatethe development of improved diagnostic reagents for both familial andacquired forms of TTP.

C. Mutants and Variants of ADAMTS13

1. Mutants of ADAMTS13 Cause Familial TTP.

DNA sequence analysis identified mutations within the ADAMTS13 gene inall 4 of the pedigrees depicted in FIG. 1, as well as in 3 additionalTTP patients not included in the original genome scan (families E-G,Table 1). These mutations are shown in Table 1.

TABLE 1 ADAMTS13 mutations in Thrombotic Thrombocytopenic Purpura (TTP).exon family nucleotide amino acid  3 B 286C > G H96D  3 E 304C > T R102C 6 E 587C > T T196I 10 D 1193G > A R398H 13 C 1582A > G R528G 13 G1584 + 5G > A splice 17 A 2074C > T R692C 19 F 2374-2399del frameshift22 B 2851T > G C951G 24 D 3070T > G C1024G 26 F 3638G > A C1213Y  26 *8 * 3655C > T * R1219W * 27 C 3769-3770insA frameshift Genomic DNA frompatients was used to amplify exons and intron/exon boundaries ofADAMTS13. For mutations in families A to D, candidate mutations wereconfirmed in both parents. Analysis of the potential splice mutation infamily G with a splice site prediction tool suggests that it shouldabolish splicing from this donor site. Consistent with this prediction,sequence analysis of PCR amplified mRNA from patient lymphoblastsidentified a major product of wild-type sequence derived only from thenormal allele. A second, slightly larger product not seen in controlsamples was derived only from the mutant allele, utilizing a crypticdonor splice site at +69, resulting in a 23 amino acid insertion.Approximately 180 normal control chromosomes were screened byallele-specific oligonucleotide hybridization, restriction digest or PCRfor the following mutations, with no mutant alleles identified: H96D,R102C, R398H, R528G, R692C, C1213Y, 2374-2399del, and 1584 + 5G > A.

An additional mutation accounting for 1 of 2 disease alleles in an 8thfamilial pedigree was also identified (indicated in Table 1 above by anasterisk (*)). Sequence analysis of exons and exon-intron junctions ofADAMTS13 was performed on genomic DNA obtained from the proband of anadditional familial TTP pedigree. The patient was found to beheterozygous for a 3655C>T substitution in exon 26. The substitution wasalso present in the heterozygous state in the affected brother andobligate carrier father, but absent in the mother and 6 unaffectedsiblings. In addition, the T allele was confirmed to be absent from 180control chromosomes by allele-specific oligonucleotide hybridization.The resulting amino acid change, R1219W, occurs within the CUB domain atthe C-terminus of ADAMTS13 (FIG. 3, panel C). No mutation was identifiedfor the other allele in this family.

The 12 mutations initially identified accounted for all but one of the15 disease alleles initially expected in this set of patients (Table 1).With the additional mutation (which accounts for 1 of 2 disease allelesin the 8^(th) family pedigree), these analyses resulted in theidentification of 15 of the 17 disease alleles in the families studied.The two unidentified mutations may lie within exon 7, or withinnoncoding regions not covered by the sequence analysis. The presence ofat least one mutation in all hereditary TTP families identified thus farindicates that most if not all cases of this disease are due tomutations in ADAMTS13. Moreover, successful identification of 15 of 17disease alleles suggests that the majority of ADAMTS13 mutations inhereditary TTP are likely to lie within the coding sequence and exerteffects on either protein stability or function.

No recurrent mutation was observed, except in family A, where all 3affected individuals are homozygous for the same mutation carried on thesame extended haplotype, suggesting a founder mutation within the SouthAmerican population of origin for this family. Two mutations result inframeshifts (a 26 bp deletion in exon 19 and single A insertion in exon27) and a single splice mutation leads to an in frame 23 amino acidinsertion. The remaining observed mutations all result innonconservative amino acid substitutions (Table 1 and followingparagraph), and all occur at positions that are perfectly conservedbetween the human and murine genes; these mutations are also locatedthroughout the length of the protein, with no apparent clustering in anyspecific domain or region of the molecule.

However, several of these mutations occur at highly conserved positionsthat could disrupt proper folding or may affect substrate binding. TheR398H mutation within the first TSP1 motif occurs at a residue that isperfectly conserved among all 18 ADAMTS family members identified todate. This mutation occurs within a conserved motif of the TSP1 domainsshown to be modified by an unusual O-linked disaccharideGlc-Fuc-O-Ser/Thr in platelet TSP1 (Hofsteenge et al. (2001) J. Biol.Chem. 276, 6485-6498) and thought to be important for ligand binding(Adams & Tucker (2000) Dev. Dyn. 218, 280-299). H96D in themetalloprotease domain occurs at a residue that is also conserved in allADAMTS family members identified to date, with the exception ofADAMTS5/11 and ADAMTS8. The R102C mutation introduces a cysteine residuewhich may disrupt a disulfide bond between C155 and C208, predictedbased on a comparison with a molecular model of adamalysin II (Zheng etal. (2001) J. Biol. Chem. 276, 41059-41063). The C951G mutation (as wellas the C1024G mutation) also affect conserved cysteine residues (Adams &Tucker (2000) Dev. Dyn. 218, 280-299; Zheng et al. (2001) J. Biol. Chem.276, 41059-41063) in the fourth and sixth TSP1 motifs of ADAMTS13,respectively. The C1213Y and R1219W mutations occur within the CUBdomain located at the C-terminus of ADAMTS13. The C1213Y mutationaffects one of several highly conserved cysteine residues within CUBdomains (http://pfam.wustl.edu) that have been proposed to formdisulfide bonds (Sieron et al. (2000) Biochemistry 39, 3231-3239). CUBdomains have been described in a number of developmentally regulatedproteins, including several zinc metalloproteases (Bork & Beckmann(1993) J. Mol. Biol. 231, 539-545); the CUB domain of BMP-1, orprocollagen-C-proteinase, has been implicated in substrate binding(Sieron et al. (2000) Biochemistry 39, 3231-3239).

The spectrum of ADAMTS13 mutations observed here is notable for therelative paucity of obvious null alleles. In addition, both frameshiftmutations are located toward the C-terminus, potentially giving rise totruncated forms of the protease that retain an intact catalytic domain.These data suggest that complete deficiency of ADAMTS13 may be lethal.This hypothesis is supported by the observed trend toward trace activityabove background seen in the majority of the mutants tested, and by thelow levels of residual VWF-cleaving protease activity observed in all 10deficient patients described here (0.02 to 0.07 U/ml).

Northern blot analysis detected an ˜4.7 kb ADAMTS13 mRNA specifically inthe liver, with a truncated, ˜2.3 kb, mRNA faintly visible in placenta(FIG. 4A). These data suggest that plasma VWF-cleaving protease may bederived primarily from ADAMTS13 expression in the liver. The strongRT-PCR signal seen in the ovary, and variable expression in othertissues (FIG. 4B), suggest other potential functions for this protein.The absence of detectable transcripts in other highly vascular tissuessuch as the lung, kidney and heart may indicate that the vascularendothelium is not a primary site of ADAMTS13 expression.

The findings reported here provide the first direct proof of anetiologic role for a VWF-cleaving protease in the pathogenesis of TTPand identify the enzyme associated with this activity as the novelmetalloproteinase ADAMTS13. These data are consistent with thehypothesis that accumulation of hyperactive large VWF multimers in theabsence of normal proteolytic processing triggers pathologic plateletaggregation and is the direct mechanism responsible for TTP.Alternatively, decreased VWF proteolysis may be a marker for the loss ofADAMTS13 activity. ADAMTS13 may also have important biologic functionselsewhere in the coagulation system or in the blood vessel wall, withloss of one or more of these activities providing the direct link to thepathogenesis of TTP.

2. ADAMTS13 Mutations in TTP Patients Result in Loss of VWF-CleavingProtease Activity.

The functional significance of the ADAMTS13 mutations identified herewas evaluated by analysis of the VWF-cleaving protease activity ofrecombinant mutant ADAMTS13. Each of the missense mutations wasengineered into the wild-type ADAMTS13 construct and transfected intoCHO-Tag cells. Analysis of VWF-cleaving protease activity in conditionedmedia revealed that all 9 mutations examined resulted in markedlydecreased activity, which is not statistically distinguishable from thatpresent in conditioned media from mock-transfected cells (FIG. 11).

Conditioned media from CHO-Tag cells transfected with the wild-type andthe missense mutant constructs were subjected to Western blot analysiswith 4 different anti-peptide antibodies raised against ADAMTS13peptides. Although one of these antibodies (antibody 4, see Materialsand Methods in the Examples) has been successfully used to detectappropriate segments of bacterially-expressed ADAMTS13, no specificfragments corresponding to the expected size of ADAMTS13 were detectablein conditioned media from cells transfected with either the wild-type ormutant constructs. In addition, epitope (FLAG)-tagged recombinantADAMTS13 was also undetectable by Western blot analysis using acommercially-available anti-FLAG antibody.

Though all 9 mutations described above exhibit marked loss ofVWF-cleaving protease activity, the loss of activity may be due tochange in protein function, synthesis, secretion, or stability. Theplasma concentration of ADAMTS13 has been estimated at ˜1 mg/ml(Gerritsen et al. (2001) Blood 98, 1654-1661). Therefore, based on theVWF-cleaving protease activity of wild-type recombinant ADAMTS13, mutantADAMTS13 is present at roughly half this concentration in therecombinant mutant ADAMTS13 samples. Although initial attempts todetermine whether mutant proteins are secreted from the cell at levelssimilar to wild-type recombinant ADAMTS13 by Western blot analysis wereunsuccessful, as described above, it is contemplated that generation ofmore sensitive antibody reagents or of epitope-tagged mutant constructswill result in such determination.

3. Variants of ADAMTS13.

A large number of SNPs were also identified, though only 7/25 result inamino acid substitutions (see Table 2 ). These SNPs all constitutenaturally occurring wild-type ADAMTS13 alleles; any particular allelemay comprise from one to more than one SNP, and different combinationsof SNPs may occur together.

TABLE 2 Single nucleotide polymorphisms exon/intron nucleotide aminoacid ex1 19C > T R7W ex4 354G > A silent ex5 420T > C silent ex6 582C >T silent int6 686 + 4T > G N/A int8 987 + 11C > T N/A int8 987 + 69C > TN/A int9 1092 + 67G > A N/A int10 1245 − 32C > G N/A ex12 1342C > GQ448E int13 1584 + 106C > G N/A int13 1584 + 236T > C N/A ex15 1716G > Asilent int15 1787 − 26G > A N/A ex16 1852C > G P618A ex16 1874G > AR625H ex18 2195C > T A732V ex19 2280T > C silent ex21 2699C > T A900Vint22 2861 + 55C > T N/A ex23 2910C > T silent ex24 3097G > A A1033Tex24 3108G > A silent int28 4077 + 32T > C N/A ex29 4221C > A silent

Of the 25 single nucleotide polymorphisms (SNPS) identified in ADAMTS13genomic sequences, 15 polymorphisms occurred within coding sequence, and7 cause amino acid substitutions. This surprising degree of polymorphismin the ADAMTS13 gene raises the possibility that one or more of theputative disease mutation identified in the initial panel of patients,though absent from 180 control chromosomes, might represent a rare“private” polymorphism within the corresponding family. However, thefunctional data shown in FIG. 11 demonstrate that all 9 mutationsdescribed above represent authentic disease mutations resulting inpartial or complete loss of ADAMTS13 function.

D. Utility of ADAMTS]3 Genes and Proteins

The present invention also provides several methods of use of wild-typeand mutants, variants and fragments of ADAMTS13 and the encodedproteins, as well as of antibodies to wild-type and mutants, variants,and fragments of ADAMTS13. In some embodiments, methods are provided forprecise and rapid diagnosis of TTP in individuals with inherited TTP.Such diagnosis is effected by any number of detection assays based uponnucleotide sequences, as described in more detail below, in which thetypes of alleles present in an individual are identified. In otherembodiments, rapid diagnosis of TTP both in the inherited and in themore common acquired form of TTP is based upon the use of antibodies todetect the presence or levels of ADAMTS13 and variants and mutants, asfor example in blood or plasma samples obtained from in individual.

The identification of ADAMTS13 deficiency as the cause of TTP also hasmajor implications for the treatment of this important human disease. Inthese embodiments, the present invention provides methods of treatingpatients with TTP. In some embodiments, a patient is administered atherapeutically effective amount of a recombinant protein. Thistreatment is likely to be much more effective, as well as much safer,than the plasma replacement therapy that is currently the onlyalternative. In yet other embodiments, a patient is treated with atherapeutically effective amount of genetic material comprising anADAMTS13 gene or mutant or variant thereof that results in production ofan ADAMTS13 protease in the patient.

In addition, ADAMTS13 or variants or other drugs based upon thisprotease can also be used in several different ways. In someembodiments, ADAMTS13 or drugs developed from it can be used in normalindividuals as a novel approach to effect anticoagulation (preventingabnormal blood clots). Since blood clots are the basis of many importanthuman diseases including heart attack and stroke, ADAMTS13 is useditself or as a suitable platform for the development of newpharmaceuticals to treat these common human diseases, where thepharmaceuticals are anticoagulants. In other embodiments, ADAMTS13 orvariants are used to deliver other therapeutic proteins specifically tothe microvasculature. These embodiments are based upon the observationthat ADAMTS13 uses VWF in a specific conformation to cleave theMet842-Tyr843 bond. This conformation is reproduced in vitro by slightly“denaturing” VWF in urea or guanidine. It is believed that such“denaturation” is achieved in vivo by shear stress in themicrovasculature. Therefore, it is contemplated that therapeuticproteins are administered in an inactive form that can be activated bycleavage of a peptide bond specifically by ADAMTS13 or variants underconditions of high shear stress in vivo.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a disintegrin and metalloproteinasecontaining thrombospondin 1-like domains (ADAMTS) and in particular to anovel ADAMTS13 protease and to nucleic acids encoding ADAMTS13proteases. The present invention encompasses both native and recombinantwild-type forms of ADAMTS13, as well as mutant and variant formsincluding fragments, some of which posses altered characteristicsrelative to the wild-type ADAMTS13. The present invention also relatesto methods of using ADAMTS13, including for treatment of TTP. Thepresent invention also relates to methods for screening for the presenceof TTP. The present invention further relates to methods for developinganticoagulant drugs based upon ADAMTS13.

I. ADAMTS13 Polynucleotides

As described above, a novel member of the family of disintegrin andmetalloproteinases containing thrombospondin 1-like domains, ADAMTS13,has been discovered. This was accomplished by studying a series offamilies in which TTP appears to be inherited and then using apositional cloning approach to map a gene responsible for reducedVWF-cleaving protease activity to a locus on 9q34. Accordingly, thepresent invention provides nucleic acids encoding ADAMTS13 genes,homologs, variants (e.g., polymorphisms and mutants), and fragments,including but not limited to, those described in SEQ ID NOs: 1, 3, 5 and7. In some embodiments, the present invention provide polynucleotidesequences that are capable of hybridizing to SEQ ID NOs: 1, 3, 5, and 7under conditions of low to high stringency as long as the polynucleotidesequence capable of hybridizing encodes a protein that retains at leastone or a portion of at least one biological activity of a naturallyoccurring ADAMTS13. In some embodiments, the protein that retains atleast one or a portion of at least one biological activity of naturallyoccurring ADAMTS13 is 70% homologous to wild-type ADAMTS13, preferably80% homologous to wild-type ADAMTS13, more preferably 90% homologous towild-type ADAMTS13, and most preferably 95% homologous to wild-typeADAMTS13. In preferred embodiments, hybridization conditions are basedon the melting temperature (T_(m)) of the nucleic acid binding complexand confer a defined “stringency” as explained above (See e.g., Wahl, etal., (1987) Meth. Enzymol., 152:399-407, incorporated herein byreference).

In other embodiments of the present invention, additional alleles ofADAMTS13 are provided. In preferred embodiments, alleles result from apolymorphism or mutation (i.e., a change in the nucleic acid sequence)and generally produce altered mRNAs or polypeptides whose structure orfunction may or may not be altered. Any given gene may have none, one ormany allelic forms. Common mutational changes that give rise to allelesare generally ascribed to deletions, additions or substitutions ofnucleic acids. Each of these types of changes may occur alone, or incombination with the others, and at the rate of one or more times in agiven sequence. Non-limiting examples of the alleles of the presentinvention include those encoded by SEQ ID NOs:1, 3, 5, and 7 (wildtype), as well as those described in Tables 1 and 2.

In some embodiments of the present invention, the nucleotide sequencesencode a CUB domain (e.g., nucleic acid sequences encoding thepolypeptide fragment from amino acid 1192 to amino acid 1286 as shown inFIG. 6).

In still other embodiments of the present invention, the nucleotidesequences of the present invention may be engineered in order to alteran ADAMTS13 coding sequence for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing and/orexpression of the gene product. For example, mutations may be introducedusing techniques that are well known in the art (e.g., site-directedmutagenesis to insert new restriction sites, to alter glycosylationpatterns, to change codon preference, etc.).

In some embodiments of the present invention, the polynucleotidesequence of ADAMTS13 may be extended utilizing the nucleotide sequences(e.g., SEQ ID NOs: 1, 3 and 7) in various methods known in the art todetect upstream sequences such as promoters and regulatory elements. Forexample, it is contemplated that restriction-site polymerase chainreaction (PCR) will find use in the present invention. This is a directmethod which uses universal primers to retrieve unknown sequenceadjacent to a known locus (Gobinda et al. (1993) PCR Methods Applic.,2:318-22). First, genomic DNA is amplified in the presence of a primerto a linker sequence and a primer specific to the known region. Theamplified sequences are then subjected to a second round of PCR with thesame linker primer and another specific primer internal to the firstone. Products of each round of PCR are transcribed with an appropriateRNA polymerase and sequenced using reverse transcriptase.

In another embodiment, inverse PCR can be used to amplify or extendsequences using divergent primers based on a known region (Triglia etal. (1988) Nucleic Acids Res., 16:8186). The primers may be designedusing Oligo 4.0 (National Biosciences Inc, Plymouth Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68-72° C. The method uses several restriction enzymesto generate a suitable fragment in the known region of a gene. Thefragment is then circularized by intramolecular ligation and used as aPCR template. In still other embodiments, walking PCR is utilized.Walking PCR is a method for targeted gene walking that permits retrievalof unknown sequence (Parker et al., (1991) Nucleic Acids Res.,19:3055-3060). The PROMOTERFINDER kit (Clontech) uses PCR, nestedprimers and special libraries to “walk in” genomic DNA. This processavoids the need to screen libraries and is useful in finding intron/exonjunctions.

Preferred libraries for screening for full length cDNAs includemammalian libraries that have been size-selected to include largercDNAs. Also, random primed libraries are preferred, in that they willcontain more sequences that contain the 5′ and upstream gene regions. Arandomly primed library may be particularly useful in case where anoligo d(T) library does not yield full-length cDNA. Genomic mammalianlibraries are useful for obtaining introns and extending 5′ sequence.

In other embodiments of the present invention, variants of the disclosedADAMTS13 sequences are provided. In preferred embodiments, variantsresult from polymorphisms or mutations (i.e., a change in the nucleicacid sequence) and generally produce altered mRNAs or polypeptides whosestructure or function may or may not be altered. Any given gene may havenone, one, or many variant forms. Non-limiting examples of variants areshown in Table 2 Common mutational changes that give rise to variantsare generally ascribed to deletions, additions or substitutions ofnucleic acids; non-limiting examples are shown in Table 1. Each of thesetypes of changes may occur alone, or in combination with the others, andat the rate of one or more times in a given sequence.

It is contemplated that it is possible to modify the structure of apeptide having a function (e.g., ADAMTS13 protease function) for suchpurposes as altering (e.g., increasing or decreasing) the substratespecificity or selectivity affinity of the ADAMTS13 for VWF or anothersubstrate. Such modified peptides are considered functional equivalentsof peptides having an activity of ADAMTS13 as defined herein. A modifiedpeptide can be produced in which the nucleotide sequence encoding thepolypeptide has been altered, such as by substitution, deletion, oraddition. In particularly preferred embodiments, these modifications donot significantly reduce the protease activity of the modified ADAMTS13.In other words, construct “X” can be evaluated in order to determinewhether it is a member of the genus of modified or variant ADAMTS13's ofthe present invention as defined functionally, rather than structurally.In preferred embodiments, the activity of variant ADAMTS13 polypeptidesis evaluated by the methods described in Example 1B. Accordingly, insome embodiments, the present invention provides nucleic acids encodinga ADAMTS13 that cleaves VWF. In preferred embodiments, the activity of aADAMTS13 variant is evaluated by utilizing guanidinehydrochloride-treated VWF.

Moreover, as described above, variant forms of ADAMTS13 and nucleotidesencoding the same are also contemplated as being equivalent to thosepeptides and DNA molecules that are set forth in more detail herein. Forexample, it is contemplated that isolated replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid (i.e., conservative mutations) will not have a majoreffect on the biological activity of the resulting molecule.Accordingly, some embodiments of the present invention provide variantsof ADAMTS13 disclosed herein containing conservative replacements.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Genetically encodedamino acids can be divided into four families: (1) acidic (aspartate,glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar(alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan); and (4) uncharged polar (glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine,tryptophan, and tyrosine are sometimes classified jointly as aromaticamino acids. In similar fashion, the amino acid repertoire can begrouped as (1) acidic (aspartate, glutamate); (2) basic (lysine,arginine, histidine), (3) aliphatic (glycine, alanine, valine, leucine,isoleucine, serine, threonine), with serine and threonine optionally begrouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine,tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine) (e.g., Stryer ed.,Biochemistry, pg. 17-21, 2nd ed, WH Freeman and Co., 1981). Whether achange in the amino acid sequence of a peptide results in a functionalpolypeptide can be readily determined by assessing the ability of thevariant peptide to function in a fashion similar to the wild-typeprotein. Peptides having more than one replacement can readily be testedin the same manner.

More rarely, a variant includes “nonconservative” changes (e.g.,replacement of a glycine with a tryptophan). Analogous minor variationscan also include amino acid deletions or insertions, or both. Guidancein determining which amino acid residues can be substituted, inserted,or deleted without abolishing biological activity can be found usingcomputer programs (e.g., LASERGENE software, DNASTAR Inc., Madison,Wis.).

As described in more detail below, variants may be produced by methodssuch as directed evolution or other techniques for producingcombinatorial libraries of variants, described in more detail below. Instill other embodiments of the present invention, the nucleotidesequences of the present invention may be engineered in order to alter aADAMTS13 coding sequence including, but not limited to, alterations thatmodify the cloning, processing, localization, secretion, and/orexpression of the gene product. Such mutations may be introduced usingtechniques that are well known in the art (e.g., site-directedmutagenesis to insert new restriction sites, alter glycosylationpatterns, or change codon preference, etc.).

II. ADAMTS13 Polypeptides

In other embodiments, the present invention provides ADAMTS13polypeptides and fragments. Non-limiting examples of ADAMTS13polypeptides (e.g., SEQ ID NOs: 2, 4 and 6) are described in FIGS. 3, 6,and 7. Other embodiments of the present invention provide fusionproteins or functional equivalents of these ADAMTS13 proteins. In stillother embodiments, the present invention provides ADAMTS13 polypeptidevariants, homologs, and mutants. In some embodiments of the presentinvention, the polypeptide is a naturally purified product, in otherembodiments it is a product of chemical synthetic procedures, and instill other embodiments it is produced by recombinant techniques using aprokaryotic or eukaryotic host (e.g., by bacterial, yeast, higher plant,insect and mammalian cells in culture). In some embodiments, dependingupon the host employed in a recombinant production procedure, thepolypeptide of the present invention may be glycosylated or it may benon-glycosylated. In other embodiments, the polypeptides of theinvention may also include an initial methionine amino acid residue.

In one embodiment of the present invention, due to the inherentdegeneracy of the genetic code, DNA sequences other than thepolynucleotide sequences of SEQ ID NO:1 and 3 which encode substantiallythe same or a functionally equivalent amino acid sequences, may be usedto clone and express ADAMTS13. In general, such polynucleotide sequenceshybridize to SEQ ID NO:1 under conditions of high to medium stringencyas described above. As will be understood by those of skill in the art,it may be advantageous to produce ADAMTS13-encoding nucleotide sequencespossessing non-naturally occurring codons. Therefore, in some preferredembodiments, codons preferred by a particular prokaryotic or eukaryotichost (Murray et al. (1989) Nucl. Acids Res. 17) are selected, forexample, to increase the rate of ADAMTS13 expression or to producerecombinant RNA transcripts having desirable properties, such as alonger half-life, than transcripts produced from naturally occurringsequence.

A. Vectors for Production of ADAMTS13

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. In some embodiments of the presentinvention, vectors include, but are not limited to, chromosomal,nonchromosomal and synthetic DNA sequences (e.g., derivatives of SV40,bacterial plasmids, phage DNA; baculovirus, yeast plasmids, vectorsderived from combinations of plasmids and phage DNA, and viral DNA suchas vaccinia, adenovirus, fowl pox virus, and pseudorabies). It iscontemplated that any vector may be used as long as it is replicable andviable in the host.

In particular, some embodiments of the present invention providerecombinant constructs comprising one or more of the sequences asbroadly described above (e.g., SEQ ID NOS: 1, 3, and 5). In someembodiments of the present invention, the constructs comprise a vector,such as a plasmid or viral vector, into which a sequence of theinvention has been inserted, in a forward or reverse orientation. Instill other embodiments, the heterologous structural sequence (e.g., SEQID NO: 1) is assembled in appropriate phase with translation initiationand termination sequences. In preferred embodiments of the presentinvention, the appropriate DNA sequence is inserted into the vectorusing any of a variety of procedures. In general, the DNA sequence isinserted into an appropriate restriction endonuclease site(s) byprocedures known in the art.

Large numbers of suitable vectors are known to those of skill in theart, and are commercially available. Such vectors include, but are notlimited to, the following vectors: 1) Bacterial—pQE70, pQE60, pQE-9(Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A,pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia); 2) Eukaryotic—pWLNEO, pSV2CAT, pOG44, PXT1,pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia); and 3)Baculovirus—pPbac and pMbac (Stratagene). Any other plasmid or vectormay be used as long as they are replicable and viable in the host. Insome preferred embodiments of the present invention, mammalianexpression vectors comprise an origin of replication, a suitablepromoter and enhancer, and also any necessary ribosome binding sites,polyadenylation sites, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking non-transcribed sequences. Inother embodiments, DNA sequences derived from the SV40 splice, andpolyadenylation sites may be used to provide the requirednon-transcribed genetic elements.

In certain embodiments of the present invention, the DNA sequence in theexpression vector is operatively linked to an appropriate expressioncontrol sequence(s) (promoter) to direct mRNA synthesis. Promotersuseful in the present invention include, but are not limited to, the LTRor SV40 promoter, the E. coli lac or trp, the phage lambda P_(L) andP_(R), T3 and T7 promoters, and the cytomegalovirus (CMV) immediateearly, herpes simplex virus (HSV) thymidine kinase, and mousemetallothionein-I promoters and other promoters known to controlexpression of gene in prokaryotic or eukaryotic cells or their viruses.In other embodiments of the present invention, recombinant expressionvectors include origins of replication and selectable markers permittingtransformation of the host cell (e.g., dihydrofolate reductase orneomycin resistance for eukaryotic cell culture, or tetracycline orampicillin resistance in E. coli).

In some embodiments of the present invention, transcription of the DNAencoding the polypeptides of the present invention by higher eukaryotesis increased by inserting an enhancer sequence into the vector.Enhancers are cis-acting elements of DNA, usually about from 10 to 300bp that act on a promoter to increase its transcription. Enhancersuseful in the present invention include, but are not limited to, theSV40 enhancer on the late side of the replication origin bp 100 to 270,a cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers.

In other embodiments, the expression vector also contains a ribosomebinding site for translation initiation and a transcription terminator.In still other embodiments of the present invention, the vector may alsoinclude appropriate sequences for amplifying expression.

B. Host Cells for Production of ADAMTS13

In a further embodiment, the present invention provides host cellscontaining the above-described constructs. In some embodiments of thepresent invention, the host cell is a higher eukaryotic cell (e.g., amammalian or insect cell). In other embodiments of the presentinvention, the host cell is a lower eukaryotic cell (e.g., a yeastcell). In still other embodiments of the present invention, the hostcell can be a prokaryotic cell (e.g., a bacterial cell). Specificexamples of host cells include, but are not limited to, Escherichiacoli, Salmonella typhimurium, Bacillus subtilis, and various specieswithin the genera Pseudomonas, Streptomyces, and Staphylococcus, as wellas Saccharomycees cerivisiae, Schizosaccharomycees pombe, Drosophila S2cells, Spodoptera Sf9 cells, Chinese hamster ovary (CHO) cells, COS-7lines of monkey kidney fibroblasts, (Gluzman, Cell 23:175 (1981)), C127,3T3, 293, 293T, HeLa and BHK cell lines, T-1 (tobacco cell cultureline), root cell and cultured roots in rhizosecretion (Gleba et al.,(1999) Proc Natl Acad Sci USA 96:5973-5977).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. In someembodiments, introduction of the construct into the host cell can beaccomplished by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (See e.g., Davis et al. (1986) BasicMethods in Molecular Biology). Alternatively, in some embodiments of thepresent invention, the polypeptides of the invention can besynthetically produced by conventional peptide synthesizers.

Proteins can be expressed in mammalian cells, yeast, bacteria, or othercells under the control of appropriate promoters. Cell-free translationsystems can also be employed to produce such proteins using RNAs derivedfrom the DNA constructs of the present invention. Appropriate cloningand expression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al. (1989) Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y.

In some embodiments of the present invention, following transformationof a suitable host strain and growth of the host strain to anappropriate cell density, the selected promoter is induced byappropriate means (e.g., temperature shift or chemical induction) andcells are cultured for an additional period. In other embodiments of thepresent invention, cells are typically harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification. In still other embodiments of thepresent invention, microbial cells employed in expression of proteinscan be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or use of cell lysingagents.

C. Purification of ADAMTS13

The present invention also provides methods for recovering and purifyingADAMTS13 from recombinant cell cultures including, but not limited to,ammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. In otherembodiments of the present invention, protein-refolding steps can beused as necessary, in completing configuration of the mature protein. Instill other embodiments of the present invention, high performanceliquid chromatography (HPLC) can be employed for final purificationsteps.

The present invention further provides polynucleotides having the codingsequence (e.g., SEQ ID NOs: 1, 3, and 5) fused in frame to a markersequence that allows for purification of the polypeptide of the presentinvention. A non-limiting example of a marker sequence is ahexahistidine tag which may be supplied by a vector, preferably a pQE-9vector, which provides for purification of the polypeptide fused to themarker in the case of a bacterial host, or, for example, the markersequence may be a hemagglutinin (HA) tag when a mammalian host (e.g.,COS-7 cells) is used. The HA tag corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al. (1984) Cell, 37:767).

D. Fragments and Domains of ADAMTS13

In addition, the present invention provides fragments of ADAMTS13 (i.e.,truncation mutants, e.g., SEQ ID NO:4). In other embodiments, thepresent invention provides domains of ADAMTS13 (e.g., the CUB domain,SEQ ID NO:6) In some embodiments of the present invention, whenexpression of a portion of the ADAMTS13 protein is desired, it may benecessary to add a start codon (ATG) to the oligonucleotide fragmentcontaining the desired sequence to be expressed. It is well known in theart that a methionine at the N-terminal position can be enzymaticallycleaved by the use of the enzyme methionine aminopeptidase (MAP). MAPhas been cloned from E. coli (Ben-Bassat et al. (1987) J. Bacteriol.,169:751) and Salmonella typhimurium and its in vitro activity has beendemonstrated on recombinant proteins (Miller et al. (1990) Proc. Natl.Acad. Sci. USA, 84:2718). Therefore, removal of an N-terminalmethionine, if desired, can be achieved either in vivo by expressingsuch recombinant polypeptides in a host which produces MAP (e.g., E.coli or CM89 or S. cerevisiae), or in vitro by use of purified MAP.

E. Fusion Proteins Containing ADAMTS13

The present invention also provides fusion proteins incorporating all orpart of ADAMTS13. Accordingly, in some embodiments of the presentinvention, the coding sequences for the polypeptide can be incorporatedas a part of a fusion gene including a nucleotide sequence encoding adifferent polypeptide. It is contemplated that this type of expressionsystem will find use under conditions where it is desirable to producean immunogenic fragment of a ADAMTS13 protein. In some embodiments ofthe present invention, the VP6 capsid protein of rotavirus is used as animmunologic carrier protein for portions of the ADAMTS13 polypeptide,either in the monomeric form or in the form of a viral particle. Inother embodiments of the present invention, the nucleic acid sequencescorresponding to the portion of ADAMTS13 against which antibodies are tobe raised can be incorporated into a fusion gene construct whichincludes coding sequences for a late vaccinia virus structural proteinto produce a set of recombinant viruses expressing fusion proteinscomprising a portion of ADAMTS13 as part of the virion. It has beendemonstrated with the use of immunogenic fusion proteins utilizing thehepatitis B surface antigen fusion proteins that recombinant hepatitis Bvirions can be utilized in this role as well. Similarly, in otherembodiments of the present invention, chimeric constructs coding forfusion proteins containing a portion of ADAMTS13 and the polioviruscapsid protein are created to enhance immunogenicity of the set ofpolypeptide antigens (See e.g., EP Publication No. 025949; and Evans etal (1989) Nature 339:385; Huang et al. (1988) J. Virol., 62:3855; andSchlienger et al. (1992) J. Virol., 66:2).

In still other embodiments of the present invention, the multipleantigen peptide system for peptide-based immunization can be utilized.In this system, a desired portion of ADAMTS13 is obtained directly fromorgano-chemical synthesis of the peptide onto an oligomeric branchinglysine core (see e.g., Posnett et al. (1988) J. Biol. Chem., 263:1719;and Nardelli et al. (1992) J. Immunol., 148:914). In other embodimentsof the present invention, antigenic determinants of the ADAMTS13proteins can also be expressed and presented by bacterial cells.

In addition to utilizing fusion proteins to enhance immunogenicity, itis widely appreciated that fusion proteins can also facilitate theexpression of proteins, such as the ADAMTS13 protein of the presentinvention. Accordingly, in some embodiments of the present invention,ADAMTS13 can be generated as a glutathione-S-transferase (i.e., GSTfusion protein). It is contemplated that such GST fusion proteins willenable easy purification of ADAMTS13, such as by the use ofglutathione-derivatized matrices (See e.g., Ausabel et al. (1992)(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY).In another embodiment of the present invention, a fusion gene coding fora purification leader sequence, such as a poly-(His)/enterokinasecleavage site sequence at the N-terminus of the desired portion ofADAMTS13, can allow purification of the expressed ADAMTS13 fusionprotein by affinity chromatography using a Ni²⁺ metal resin. In stillanother embodiment of the present invention, the purification leadersequence can then be subsequently removed by treatment with enterokinase(See e.g., Hochuli et al. (1987) J. Chromatogr., 411:177; and Janknechtet al., Proc. Natl. Acad. Sci. USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment ofthe present invention, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, in other embodiments of the present invention, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed to generate a chimeric genesequence (See e.g., Current Protocols in Molecular Biology, supra).

F. Variants of ADAMTS13

Still other embodiments of the present invention provide mutant orvariant forms of ADMTS13 (i.e., muteins; see for example Table 1). It ispossible to modify the structure of a peptide having an activity ofADAMTS13 for such purposes as enhancing therapeutic or prophylacticefficacy, or stability (e.g., ex vivo shelf life, and/or resistance toproteolytic degradation in vivo). Such modified peptides are consideredfunctional equivalents of peptides having an activity of the subjectADAMTS13 proteins as defined herein. A modified peptide can be producedin which the amino acid sequence has been altered, such as by amino acidsubstitution, deletion, or addition.

Moreover, as described above, variant forms (e.g., mutants orpolymorphic sequences) of the subject ADAMTS13 proteins and thenucleotides encoding them are also contemplated as being equivalent tothose peptides and DNA molecules that are set forth in more detail. Forexample, as described above, the present invention encompasses mutantand variant proteins that contain conservative or non-conservative aminoacid substitutions.

This invention further contemplates a method of generating sets ofcombinatorial mutants of the present ADAMTS13 proteins, as well astruncation mutants, and is especially useful for identifying potentialvariant sequences (i.e., mutants or polymorphic sequences) that arefunctional in cleaving VWF proteins or other protein substrates. Thepurpose of screening such combinatorial libraries is to generate, forexample, novel ADAMTS13 variants that can act as anticoagulants.

Therefore, in some embodiments of the present invention, ADAMTS13variants are engineered by the present method to provide alteredsubstrate specificity or selectivity. In other embodiments of thepresent invention, combinatorially-derived variants are generated whichhave a selective potency relative to a naturally occurring ADAMTS13.Such proteins, when expressed from recombinant DNA constructs, can beused in gene therapy protocols.

Still other embodiments of the present invention provide ADAMTS13variants that have intracellular half-lives dramatically different thanthe corresponding wild-type protein. For example, the altered proteincan be rendered either more stable or less stable to proteolyticdegradation or other cellular process that result in destruction of, orotherwise inactivate ADAMTS13. Such variants, and the genes which encodethem, can be utilized to alter the location of ADAMTS13 expression bymodulating the half-life of the protein. For instance, a short half-lifecan give rise to more transient ADAMTS13 biological effects and, whenpart of an inducible expression system, can allow tighter control ofADAMTS13 levels within the cell. As above, such proteins, andparticularly their recombinant nucleic acid constructs, can be used ingene therapy protocols.

In some embodiments of the combinatorial mutagenesis approach of thepresent invention, the amino acid sequences for a population of ADAMTS13homologs, variants or other related proteins are aligned, preferably topromote the highest homology possible. Such a population of variants caninclude, for example, ADAMTS13 homologs from one or more species, orADAMTS13 variants from the same species but which differ due to mutationor polymorphisms. Amino acids that appear at each position of thealigned sequences are selected to create a degenerate set ofcombinatorial sequences.

In a preferred embodiment of the present invention, the combinatorialADAMTS13 library is produced by way of a degenerate library of genesencoding a library of polypeptides which each include at least a portionof potential ADAMTS13 protein sequences. For example, a mixture ofsynthetic oligonucleotides can be enzymatically ligated into genesequences such that the degenerate set of potential ADAMTS13 sequencesare expressible as individual polypeptides, or alternatively, as a setof larger fusion proteins (e.g., for phage display) containing the setof ADAMTS13 sequences therein.

There are many ways by which the library of potential ADAMTS13 homologsand variants can be generated from a degenerate oligonucleotidesequence. In some embodiments, chemical synthesis of a degenerate genesequence is carried out in an automatic DNA synthesizer, and thesynthetic genes are ligated into an appropriate gene for expression. Thepurpose of a degenerate set of genes is to provide, in one mixture, allof the sequences encoding the desired set of potential ADAMTS13sequences. The synthesis of degenerate oligonucleotides is well known inthe art (See e.g., Narang (1983) Tetrahedron Lett., 39:39; Itakura etal. (1981) Recombinant DNA, in Walton (ed.), Proceedings of the 3rdCleveland Symposium on Macromolecules, Elsevier, Amsterdam, pp 273-289;Itakura et al. (1984) Annu. Rev. Biochem., 53:323; Itakura et al. (1984)Science 198:1056; Ike et al. (1983) Nucl. Acid Res., 11:477). Suchtechniques have been employed in the directed evolution of otherproteins (See e.g., Scott et al. (1980) Science 249:386; Roberts et al.(1992) Proc. Natl. Acad. Sci. USA 89:2429; Devlin et al. (1990) Science249: 404; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6378; aswell as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815; each ofwhich is incorporated herein by reference).

It is contemplated that the ADAMTS13 encoding nucleic acids (e.g., SEQID NO:1 and 3, and fragments and variants thereof) can be utilized asstarting nucleic acids for directed evolution. These techniques can beutilized to develop ADAMTS13 variants having desirable properties suchas increased or decreased specificity for VWF or other proteinsubstrates.

In some embodiments, artificial evolution is performed by randommutagenesis (e.g., by utilizing error-prone PCR to introduce randommutations into a given coding sequence). This method requires that thefrequency of mutation be finely tuned. As a general rule, beneficialmutations are rare, while deleterious mutations are common. This isbecause the combination of a deleterious mutation and a beneficialmutation often results in an inactive enzyme. The ideal number of basesubstitutions for targeted gene is usually between 1.5 and 5 (Moore andArnold (1996) Nat. Biotech., 14, 458; Leung et al. (1989) Technique,1:11; Eckert and Kunkel (1991) PCR Methods Appl., 1: 17-24; Caldwell andJoyce (1992) PCR Methods Appl., 2:28; and Zhao and Arnold (1997) Nuc.Acids. Res., 25:1307). After mutagenesis, the resulting clones areselected for desirable activity (e.g., screened for ADAMTS13 activity).Successive rounds of mutagenesis and selection are often necessary todevelop enzymes with desirable properties. It should be noted that onlythe useful mutations are carried over to the next round of mutagenesis.

In other embodiments of the present invention, the polynucleotides ofthe present invention are used in gene shuffling or sexual PCRprocedures (e.g., Smith (1994) Nature, 370:324; U.S. Pat. Nos.5,837,458; 5,830,721; 5,811,238; 5,733,731; all of which are hereinincorporated by reference). Gene shuffling involves random fragmentationof several mutant DNAs followed by their reassembly by PCR into fulllength molecules. Examples of various gene shuffling procedures include,but are not limited to, assembly following DNase treatment, thestaggered extension process (STEP), and random priming in vitrorecombination. In the DNase mediated method, DNA segments isolated froma pool of positive mutants are cleaved into random fragments with DNaseIand subjected to multiple rounds of PCR with no added primer. Thelengths of random fragments approach that of the uncleaved segment asthe PCR cycles proceed, resulting in mutations in present in differentclones becoming mixed and accumulating in some of the resultingsequences. Multiple cycles of selection and shuffling have led to thefunctional enhancement of several enzymes (Stemmer (1994) Nature,370:398; Stemmer (1994) Proc. Natl. Acad. Sci. USA, 91:10747; Crameri etal. (1996) Nat. Biotech., 14:315; Zhang et al. (1997) Proc. Natl. Acad.Sci. USA, 94:4504; and Crameri et al. (1997) Nat. Biotech., 15:436).Variants produced by directed evolution can be screened for ADAMTS13activity by the methods described in Example 1B.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations, and forscreening cDNA libraries for gene products having a certain property.Such techniques will be generally adaptable for rapid screening of thegene libraries generated by the combinatorial mutagenesis orrecombination of ADAMTS13 homologs or variants. The most widely usedtechniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected.

G. Chemical Synthesis of ADAMTS13

In an alternate embodiment of the invention, the coding sequence ofADAMTS13 is synthesized, whole or in part, using chemical methods wellknown in the art (See e.g., Caruthers et al. (1980) Nucl. Acids Res.Symp. Ser., 7:215; Crea and Horn (1980) Nucl. Acids Res., 9:2331;Matteucci and Caruthers (1980) Tetrahedron Lett., 21:719; and Chow andKempe (1981) Nucl. Acids Res., 9:2807). In other embodiments of thepresent invention, the protein itself is produced using chemical methodsto synthesize either an entire ADAMTS13 amino acid sequence or a portionthereof. For example, peptides can be synthesized by solid phasetechniques, cleaved from the resin, and purified by preparative highperformance liquid chromatography (See e.g., Creighton (1983) ProteinsStructures And Molecular Principles, W H Freeman and Co, New York N.Y.).In other embodiments of the present invention, the composition of thesynthetic peptides is confirmed by amino acid analysis or sequencing(See e.g., Creighton, supra).

Direct peptide synthesis can be performed using various solid-phasetechniques (Roberge et al. (1995) Science 269:202) and automatedsynthesis may be achieved, for example, using ABI 431A PeptideSynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer. Additionally, the amino acid sequence of ADAMTS13,or any part thereof, may be altered during direct synthesis and/orcombined using chemical methods with other sequences to produce avariant polypeptide.

III. Detection of ADAMTS13 Alleles

A. ADAMTS13 Alleles

In some embodiments, the present invention includes alleles of ADAMTS13that increase a patient's susceptibility to TTP disease (e.g.,including, but not limited to, the mutations shown in Table 1). Analysisof naturally occurring human ADAMTS13 alleles revealed that patientswith increased susceptibility to TTP disease have a mutant ADAMTS13allele that, for example, result in a frameshift (a 26 bp deletion inexon 19, 2374-2399del, and a single A insertion in exon 27,3769-3770insA), an in frame 23 amino acid insertion as result of asingle splice mutation (1584+5>A), or a non-conservative amino acidsubstitution (286G>G, H96D; 304 C>T, R102C; 587C>T, T1961; 1193G>A,R398H; 1582A>G, R528G; 2074C>T; R692C; 2851T>G, C951G; 3070T>G, C1024G;3638G>A, C1213Y). These patients all have greatly decreased levels ofVWF-cleaving protease levels (see FIG. 1).

The present invention is not limited to a particular mechanism ofaction. Indeed, an understanding of the mechanism of action is notnecessary to practice the present invention. Nevertheless, it iscontemplated that ADAMTS13 is involved in normal proteolytic processingof VWF. It is contemplated that in TTP the accumulation of hyperactivelarge VWF multimers in the absence of normal proteolytic processingtriggers pathologic platelet aggregation and is the direct mechanismresponsible for TTP.

However, the present invention is not limited to the mutations describedin Table 1. Any mutation that results in the undesired phenotype (e.g.,a low level of VWF cleaving protease activity, or the presence of orsusceptibility to TTP) is within the scope of the present invention.Assays for determining if a given polypeptide has a decreased level ofVWF cleaving protease activity are provided in Example 1C.

For example, in some embodiments, the present invention provides allelescontaining one or more single-nucleotide changes of ADAMTS13 (e.g.,mutants or polymorphic sequences) (e.g., including but not limited tothe mutations shown in Table 1, and the polymorphisms shown in Table 2).

B. Detection of Variant Alleles

Accordingly, the present invention provides methods for determiningwhether a patient has an increased susceptibility to TTP disease bydetermining whether the individual has a variant ADAMTS13 allele. Inother embodiments, the present invention provides methods for providinga prognosis of increased risk for TTP disease to an individual based onthe presence or absence of one or more variant alleles of ADAMTS13. Inpreferred embodiments, the variation is a mutation resulting indecreased levels of VWF cleaving protease activity. In more preferredembodiments, the variation is a mutation described in Table 1.

A number of methods are available for analysis of variant (e.g., mutantor polymorphic) nucleic acid sequences. Assays for detectionspolymorphisms or mutations fall into several categories, including, butnot limited to direct sequencing assays, fragment polymorphism assays,hybridization assays, and computer based data analysis. Protocols andcommercially available kits or services for performing multiplevariations of these assays are available. In some embodiments, assaysare performed in combination or in hybrid (e.g., different reagents ortechnologies from several assays are combined to yield one assay). Thefollowing assays are useful in the present invention.

1. Direct Sequencing Assays

In some embodiments of the present invention, variant sequences aredetected using a direct sequencing technique. In these assays, DNAsamples are first isolated from a subject using any suitable method. Insome embodiments, the region of interest is cloned into a suitablevector and amplified by growth in a host cell (e.g., a bacteria). Inother embodiments, DNA in the region of interest is amplified using PCR.

Following amplification, DNA in the region of interest (e.g., the regioncontaining the SNP or mutation of interest) is sequenced using anysuitable method, including but not limited to manual sequencing usingradioactive marker nucleotides, or automated sequencing. The results ofthe sequencing are displayed using any suitable method. The sequence isexamined and the presence or absence of a given SNP or mutation isdetermined.

2. PCR Assays

In some embodiments of the present invention, variant sequences aredetected using a PCR-based assay. In some embodiments, the PCR assaycomprises the use of oligonucleotide primers that hybridize only to thevariant or wild type allele of ADAMTS13 (e.g., to the region ofpolymorphism or mutation). Both sets of primers are used to amplify asample of DNA. If only the mutant primers result in a PCR product, thenthe patient has the mutant ADAMTS13 allele. If only the wild-typeprimers result in a PCR product, then the patient has the wild typeallele of ADAMTS13.

3. Fragment Length Polymorphism Assays

In some embodiments of the present invention, variant sequences aredetected using a fragment length polymorphism assay. In a fragmentlength polymorphism assay, a unique DNA banding pattern based oncleaving the DNA at a series of positions is generated using an enzyme(e.g., a restriction enzyme or a CLEAVASE I (Third Wave Technologies,Madison, Wis.) enzyme). DNA fragments from a sample containing a SNP ora mutation will have a different banding pattern than wild type.

a. RFLP Assays

In some embodiments of the present invention, variant sequences aredetected using a restriction fragment length polymorphism assay (RFLP).The region of interest is first isolated using PCR. The PCR products arethen cleaved with restriction enzymes known to give a unique lengthfragment for a given polymorphism. The restriction-enzyme digested PCRproducts are separated by agarose gel electrophoresis and visualized byethidium bromide staining. The length of the fragments is compared tomolecular weight markers and fragments generated from wild-type andmutant controls.

b. CFLP Assays

In other embodiments, variant sequences are detected using a CLEAVASEfragment length polymorphism assay (CFLP; Third Wave Technologies,Madison, Wis.; See e.g., U.S. Pat. Nos. 5,843,654; 5,843,669; 5,719,208;and 5,888,780; each of which is herein incorporated by reference). Thisassay is based on the observation that when single strands of DNA foldon themselves, they assume higher order structures that are highlyindividual to the precise sequence of the DNA molecule. These secondarystructures involve partially duplexed regions of DNA such that singlestranded regions are juxtaposed with double stranded DNA hairpins. TheCLEAVASE I enzyme, is a structure-specific, thermostable nuclease thatrecognizes and cleaves the junctions between these single-stranded anddouble-stranded regions.

The region of interest is first isolated, for example, using PCR. Then,DNA strands are separated by heating. Next, the reactions are cooled toallow intrastrand secondary structure to form. The PCR products are thentreated with the CLEAVASE I enzyme to generate a series of fragmentsthat are unique to a given SNP or mutation. The CLEAVASE enzyme treatedPCR products are separated and detected (e.g., by agarose gelelectrophoresis) and visualized (e.g., by ethidium bromide staining).The length of the fragments is compared to molecular weight markers andfragments generated from wild-type and mutant controls.

4. Hybridization Assays

In preferred embodiments of the present invention, variant sequences aredetected by hybridization analysis in a hybridization assay. In ahybridization assay, the presence of absence of a given SNP or mutationis determined based on the ability of the DNA from the sample tohybridize to a complementary DNA molecule (e.g., a oligonucleotideprobe). A variety of hybridization assays using a variety oftechnologies for hybridization and detection are available. Adescription of a selection of assays is provided below.

a. Direct Detection of Hybridization

In some embodiments, hybridization of a probe to the sequence ofinterest (e.g., a SNP or mutation) is detected directly by visualizing abound probe (e.g., a Northern or Southern assay; See e.g., Ausabel etal. (eds.) (1991) Current Protocols in Molecular Biology, John Wiley &Sons, NY). In a these assays, genomic DNA (Southern) or RNA (Northern)is isolated from a subject. The DNA or RNA is then cleaved with a seriesof restriction enzymes that cleave infrequently in the genome and notnear any of the markers being assayed. The DNA or RNA is then separated(e.g., on an agarose gel) and transferred to a membrane. A labeled(e.g., by incorporating a radionucleotide) probe or probes specific forthe SNP or mutation being detected is allowed to contact the membraneunder a condition or low, medium, or high stringency conditions. Unboundprobe is removed and the presence of binding is detected by visualizingthe labeled probe.

b. Detection of Hybridization Using “DNA Chip” Assays

In some embodiments of the present invention, variant sequences aredetected using a DNA chip hybridization assay. In this assay, a seriesof oligonucleotide probes are affixed to a solid support. Theoligonucleotide probes are designed to be unique to a given SNP ormutation. The DNA sample of interest is contacted with the DNA “chip”and hybridization is detected.

In some embodiments, the DNA chip assay is a GeneChip (Affymetrix, SantaClara, Calif,; See e.g., U.S. Pat. Nos. 6,045,996; 5,925,525; and5,858,659; each of which is herein incorporated by reference) assay. TheGeneChip technology uses miniaturized, high-density arrays ofoligonucleotide probes affixed to a “chip.” Probe arrays aremanufactured by Affymetrix's light-directed chemical synthesis process,which combines solid-phase chemical synthesis with photolithographicfabrication techniques employed in the semiconductor industry. Using aseries of photolithographic masks to define chip exposure sites,followed by specific chemical synthesis steps, the process constructshigh-density arrays of oligonucleotides, with each probe in a predefinedposition in the array. Multiple probe arrays are synthesizedsimultaneously on a large glass wafer. The wafers are then diced, andindividual probe arrays are packaged in injection-molded plasticcartridges, which protect them from the environment and serve aschambers for hybridization.

The nucleic acid to be analyzed is isolated, amplified by PCR, andlabeled with a fluorescent reporter group. The labeled DNA is thenincubated with the array using a fluidics station. The array is theninserted into the scanner, where patterns of hybridization are detected.The hybridization data are collected as light emitted from thefluorescent reporter groups already incorporated into the target, whichis bound to the probe array. Probes that perfectly match the targetgenerally produce stronger signals than those that have mismatches.Since the sequence and position of each probe on the array are known, bycomplementarity, the identity of the target nucleic acid applied to theprobe array can be determined.

In other embodiments, a DNA microchip containing electronically capturedprobes (Nanogen, San Diego, Calif.) is utilized (See e.g., U.S. Pat.Nos. 6,017,696; 6,068,818; and 6,051,380; each of which are hereinincorporated by reference). Through the use of microelectronics,Nanogen's technology enables the active movement and concentration ofcharged molecules to and from designated test sites on its semiconductormicrochip. DNA capture probes unique to a given SNP or mutation areelectronically placed at, or “addressed” to, specific sites on themicrochip. Since DNA has a strong negative charge, it can beelectronically moved to an area of positive charge.

First, a test site or a row of test sites on the microchip iselectronically activated with a positive charge. Next, a solutioncontaining the DNA probes is introduced onto the microchip. Thenegatively charged probes rapidly move to the positively charged sites,where they concentrate and are chemically bound to a site on themicrochip. The microchip is then washed and another solution of distinctDNA probes is added until the array of specifically bound DNA probes iscomplete.

A test sample is then analyzed for the presence of target DNA moleculesby determining which of the DNA capture probes hybridize, withcomplementary DNA in the test sample (e.g., a PCR amplified gene ofinterest). An electronic charge is also used to move and concentratetarget molecules to one or more test sites on the microchip. Theelectronic concentration of sample DNA at each test site promotes rapidhybridization of sample DNA with complementary capture probes(hybridization may occur in minutes). To remove any unbound ornonspecifically bound DNA from each site, the polarity or charge of thesite is reversed to negative, thereby forcing any unbound ornonspecifically bound DNA back into solution away from the captureprobes. A laser-based fluorescence scanner is used to detect binding,

In still further embodiments, an array technology based upon thesegregation of fluids on a flat surface (chip) by differences in surfacetension (ProtoGene, Palo Alto, Calif.) is utilized (See e.g., U.S. Pat.Nos. 6,001,311; 5,985,551; and 5,474,796; each of which is hereinincorporated by reference). Protogene's technology is based on the factthat fluids can be segregated on a flat surface by differences insurface tension that have been imparted by chemical coatings. Once sosegregated, oligonucleotide probes are synthesized directly on the chipby ink-jet printing of reagents. The array with its reaction sitesdefined by surface tension is mounted on a X/Y translation stage under aset of four piezoelectric nozzles, one for each of the four standard DNAbases. The translation stage moves along each of the rows of the arrayand the appropriate reagent is delivered to each of the reaction site.For example, the amidite A is delivered only to the sites where amiditeA is to be coupled during that synthesis step and so on. Common reagentsand washes are delivered by flooding the entire surface and thenremoving them by spinning.

DNA probes unique for the SNP or mutation of interest are affixed to thechip using Protogene's technology. The chip is then contacted with thePCR-amplified genes of interest. Following hybridization, unbound DNA isremoved and hybridization is detected using any suitable method (e.g.,by fluorescence de-quenching of an incorporated fluorescent group).

In yet other embodiments, a “bead array” is used for the detection ofpolymorphisms (Illumina, San Diego, Calif.; See e.g., PCT PublicationsWO 99/67641 and WO 00/39587, each of which is herein incorporated byreference). Illumina uses a BEAD ARRAY technology that combines fiberoptic bundles and beads that self-assemble into an array. Each fiberoptic bundle contains thousands to millions of individual fibersdepending on the diameter of the bundle. The beads are coated with anoligonucleotide specific for the detection of a given SNP or mutation.Batches of beads are combined to form a pool specific to the array. Toperform an assay, the BEAD ARRAY is contacted with a prepared subjectsample (e.g., DNA). Hybridization is detected using any suitable method.

c. Enzymatic Detection of Hybridization

In some embodiments of the present invention, hybridization is detectedby enzymatic cleavage of specific structures (INVADER assay, Third WaveTechnologies; See e.g., U.S. Pat. Nos. 5,846,717, 6,090,543; 6,001,567;5,985,557; and 5,994,069; each of which is herein incorporated byreference). The INVADER assay detects specific DNA and RNA sequences byusing structure-specific enzymes to cleave a complex formed by thehybridization of overlapping oligonucleotide probes. Elevatedtemperature and an excess of one of the probes enable multiple probes tobe cleaved for each target sequence present without temperature cycling.These cleaved probes then direct cleavage of a second labeled probe. Thesecondary probe oligonucleotide can be 5′-end labeled with fluoresceinthat is quenched by an internal dye. Upon cleavage, the de-quenchedfluorescein labeled product may be detected using a standardfluorescence plate reader.

The INVADER assay detects specific mutations and SNPs in unamplifiedgenomic DNA. The isolated DNA sample is contacted with the first probespecific either for a SNP/mutation or wild type sequence and allowed tohybridize. Then a secondary probe, specific to the first probe, andcontaining the fluorescein label, is hybridized and the enzyme is added.Binding is detected by using a fluorescent plate reader and comparingthe signal of the test sample to known positive and negative controls.

In some embodiments, hybridization of a bound probe is detected using aTaqMan assay (PE Biosystems, Foster City, Calif.; See e.g., U.S. Pat.Nos. 5,962,233 and 5,538,848, each of which is herein incorporated byreference). The assay is performed during a PCR reaction. The TaqManassay exploits the 5′-3′ exonuclease activity of the AMPLITAQ GOLD DNApolymerase. A probe, specific for a given allele or mutation, isincluded in the PCR reaction. The probe consists of an oligonucleotidewith a 5′-reporter dye (e.g., a fluorescent dye) and a 3′-quencher dye.During PCR, if the probe is bound to its target, the 5′-3′ nucleolyticactivity of the AMPLITAQ GOLD polymerase cleaves the probe between thereporter and the quencher dye. The separation of the reporter dye fromthe quencher dye results in an increase of fluorescence. The signalaccumulates with each cycle of PCR and can be monitored with afluorimeter.

In still further embodiments, polymorphisms are detected using theSNP-IT primer extension assay (Orchid Biosciences, Princeton, N.J.; Seee.g., U.S. Pat. Nos. 5,952,174 and 5,919,626, each of which is hereinincorporated by reference). In this assay, SNPs are identified by usinga specially synthesized DNA primer and a DNA polymerase to selectivelyextend the DNA chain by one base at the suspected SNP location. DNA inthe region of interest is amplified and denatured. Polymerase reactionsare then performed using miniaturized systems called microfluidics.Detection is accomplished by adding a label to the nucleotide suspectedof being at the SNP or mutation location. Incorporation of the labelinto the DNA can be detected by any suitable method (e.g., if thenucleotide contains a biotin label, detection is via a fluorescentlylabeled antibody specific for biotin).

5. Mass Spectroscopy Assays

In some embodiments, a MassARRAY system (Sequenom, San Diego, Calif.) isused to detect variant sequences (See e.g., U.S. Pat. Nos. 6,043,031;5,777,324; and 5,605,798; each of which is herein incorporated byreference). DNA is isolated from blood samples using standardprocedures. Next, specific DNA regions containing the mutation or SNP ofinterest, about 200 base pairs in length, are amplified by PCR. Theamplified fragments are then attached by one strand to a solid surfaceand the non-immobilized strands are removed by standard denaturation andwashing. The remaining immobilized single strand then serves as atemplate for automated enzymatic reactions that produce genotypespecific diagnostic products.

Very small quantities of the enzymatic products, typically five to tennanoliters, are then transferred to a SpectroCHIP array for subsequentautomated analysis with the SpectroREADER mass spectrometer. Each spotis preloaded with light absorbing crystals that form a matrix with thedispensed diagnostic product. The MassARRAY system uses MALDI-TOF(Matrix Assisted Laser Desorption Ionization-Time of Flight) massspectrometry. In a process known as desorption, the matrix is hit with apulse from a laser beam. Energy from the laser beam is transferred tothe matrix and it is vaporized resulting in a small amount of thediagnostic product being expelled into a flight tube. As the diagnosticproduct is charged, when an electrical field pulse is subsequentlyapplied to the tube the diagnostic product is launched down the flighttube towards a detector. The time between application of the electricalfield pulse and collision of the diagnostic product with the detector isreferred to as the time of flight. This is a very precise measure of theproduct's molecular weight, as a molecule's mass correlates directlywith time of flight with smaller molecules flying faster than largermolecules. The entire assay is completed in less than one thousandth ofa second, enabling samples to be analyzed in a total of 3-5 secondincluding repetitive data collection. The SpectroTYPER software thencalculates, records, compares and reports the genotypes at the rate ofthree seconds per sample.

6. Variant Analysis by Differential Antibody Binding

In other embodiments of the present invention, antibodies (See below forantibody production) are used to determine if an individual contains anallele encoding an ADAMTS13 gene containing a mutation. In preferredembodiments, antibodies are utilized that discriminate between mutant(i.e., truncated proteins); and wild-type proteins (SEQ ID NO:2).

7. Kits for Analyzing Risk of TTP Disease

The present invention also provides kits for determining whether anindividual contains a wild-type or variant (e.g., mutant or polymorphic)allele of ADAMTS13. In some embodiments, the kits are useful determiningwhether the subject is at risk of developing TTP disease. The diagnostickits are produced in a variety of ways. In some embodiments, the kitscontain at least one reagent for specifically detecting a mutantADAMTS13 allele or protein. In some preferred embodiments, the kitscontain reagents for detecting a SNP caused by a single nucleotidesubstitution of the wild-type gene. In these preferred embodiments, thereagent is a nucleic acid that hybridizes to nucleic acids containingthe SNP and that does not bind to nucleic acids that do not contain theSNP. In other preferred embodiments, the reagents are primers foramplifying the region of DNA containing the SNP. In still otherembodiments, the reagents are antibodies that preferentially bind eitherthe wild-type or mutant ADAMTS13 proteins. In some embodiments, the kitcontains instructions for determining whether the subject is at risk fordeveloping TTP disease. In preferred embodiments, the instructionsspecify that risk for developing TTP disease is determined by detectingthe presence or absence of a mutant ADAMTS13 allele in the subject,wherein subjects having an allele containing a single nucleotidesubstitution mutation have an increased risk of developing TTP disease.In some embodiments, the kits include ancillary reagents such asbuffering agents, nucleic acid stabilizing reagents, protein stabilizingreagents, and signal producing systems (e.g., florescence generatingsystems as Fret systems). The test kit may be packages in any suitablemanner, typically with the elements in a single container or variouscontainers as necessary along with a sheet of instructions for carryingout the test. In some embodiments, the kits also preferably include apositive control sample.

8. Bioinformatics

In some embodiments, the present invention provides methods ofdetermining an individual's risk of developing TTP disease based on thepresence of one or more variant alleles of ADAMTS13. In someembodiments, the analysis of variant data is processed by a computerusing information stored on a computer (e.g., in a database). Forexample, in some embodiments, the present invention provides abioinformatics research system comprising a plurality of computersrunning a multi-platform object oriented programming language (See e.g.,U.S. Pat. No. 6,125,383; herein incorporated by reference). In someembodiments, one of the computers stores genetics data (e.g., the riskof contacting TTP disease associated with a given polymorphism, as wellas the sequences). Results are then delivered to the user (e.g., via oneof the computers or via the internet).

IV. Generation of ADAMTS13 Antibodies

Antibodies can be generated to allow for the detection of ADAMTS13protein. The antibodies may be prepared using various immunogens. In oneembodiment, the immunogen is an ADAMTS13 peptide to generate antibodiesthat recognize human ADAMTS13. Such antibodies include, but are notlimited to polyclonal, monoclonal, chimeric, single chain, Fabfragments, and Fab expression libraries.

Various procedures known in the art may be used for the production ofpolyclonal antibodies directed against ADAMTS13. For the production ofantibody, various host animals can be immunized by injection with thepeptide corresponding to the ADAMTS13 epitope including but not limitedto rabbits, mice, rats, sheep, goats, etc. In a preferred embodiment,the peptide is conjugated to an immunogenic carrier (e.g., diphtheriatoxoid, bovine serum albumin (BSA), or keyhole limpet hemocyanin (KLH)).Various adjuvants may be used to increase the immunological response,depending on the host species, including but not limited to Freund's(complete and incomplete), mineral gels (e.g., aluminum hydroxide),surface active substances (e.g., lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(Bacille Calmette-Guerin) and Corynebacterium parvum).

For preparation of monoclonal antibodies directed toward ADAMTS13, it iscontemplated that any technique that provides for the production ofantibody molecules by continuous cell lines in culture will find usewith the present invention (See e.g., Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.). These include but are not limited to the hybridomatechnique originally developed by Köhler and Milstein (Köhler & Milstein(1975) Nature 256:495-497), as well as the trioma technique, the humanB-cell hybridoma technique (See e.g., Kozbor et al. (1983) Immunol.Tod., 4:72), and the EBV-hybridoma technique to produce human monoclonalantibodies (Cole et al. (1985) in Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-6).

In an additional embodiment of the invention, monoclonal antibodies areproduced in germ-free animals utilizing technology such as thatdescribed in PCT/US90/02545). Furthermore, it is contemplated that humanantibodies will be generated by human hybridomas (Cote et al. (1983)Proc. Natl. Acad. Sci. USA 80:2026-2030) or by transforming human Bcells with EBV virus in vitro (Cole et al. (1985) in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).

In addition, it is contemplated that techniques described for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778; hereinincorporated by reference) will find use in producing ADAMTS13 specificsingle chain antibodies. An additional embodiment of the inventionutilizes the techniques described for the construction of Fab expressionlibraries (Huse et al. (1989) Science 246:1275-1281) to allow rapid andeasy identification of monoclonal Fab fragments with the desiredspecificity for ADAMTS13.

It is contemplated that any technique suitable for producing antibodyfragments will find use in generating antibody fragments that containthe idiotype (antigen binding region) of the antibody molecule. Forexample, such fragments include but are not limited to: F(ab′)2 fragmentthat can be produced by pepsin digestion of the antibody molecule; Fab′fragments that can be generated by reducing the disulfide bridges of theF(ab′)2 fragment, and Fab fragments that can be generated by treatingthe antibody molecule with papain and a reducing agent.

In the production of antibodies, it is contemplated that screening forthe desired antibody will be accomplished by techniques known in the art(e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay),“sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitation reactions, immunodiffusion assays, in situ immunoassays(e.g., using colloidal gold, enzyme or radioisotope labels, forexample), Western blots, precipitation reactions, agglutination assays(e.g., gel agglutination assays, hemagglutination assays, etc.),complement fixation assays, immunofluorescence assays, protein A assays,and immunoelectrophoresis assays, etc.

In one embodiment, antibody binding is detected by detecting a label onthe primary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled. Many means are known in the art for detecting binding in animmunoassay and are within the scope of the present invention. As iswell known in the art, the immunogenic peptide should be provided freeof the carrier molecule used in any immunization protocol. For example,if the peptide was conjugated to KLH, it may be conjugated to BSA, orused directly, in a screening assay.)

The foregoing antibodies can be used in methods known in the artrelating to the localization and structure of ADAMTS13 (e.g., forWestern blotting), measuring levels thereof in appropriate biologicalsamples, etc. The antibodies can be used to detect ADAMTS13 in abiological sample from an individual. The biological sample can be abiological fluid, such as, but not limited to, blood, serum, plasma,interstitial fluid, urine, cerebrospinal fluid, and the like, containingcells.

The biological samples can then be tested directly for the presence ofADAMTS13 using an appropriate strategy (e.g., ELISA or radioimmunoassay)and format (e.g., microwells, dipstick (e.g., as described inInternational Patent Publication WO 93/03367), etc. Alternatively,proteins in the sample can be size separated (e.g., by polyacrylamidegel electrophoresis (PAGE), in the presence or not of sodium dodecylsulfate (SDS), and the presence of ADAMTS13 detected by immunoblotting(Western blotting). Immunoblotting techniques are generally moreeffective with antibodies generated against a peptide corresponding toan epitope of a protein, and hence, are particularly suited to thepresent invention.

In other embodiments, the antigen is a peptide fragment of ADAMTS13;preferably, the fragment is of high antigenicity. In yet otherembodiment, the immunogen is a variant or mutant of ADAMTS13 peptide togenerate antibodies that recognize the variant or mutant ADAMTS13. Suchantibodies include, but are not limited to polyclonal, monoclonal,chimeric, single chain, Fab fragments, and Fab expression libraries, andare prepared and used as described above. These antibodies can then beused to detect the presence of a fragment or variant or mutant ADAMTS13in a biological sample from an individual, as described above, and thusto predict the susceptibility of the individual to TTP.

For example, peptide antibodies have been synthesized against onepeptide in exon 5 and one peptide in exon 13. These peptide fragmentswere selected on the basis of determinations by computer algorithms andother methods as having high “antigenicity” (likely to elicit an immuneresponse); the selected peptides were then synthesized. The peptidefragments were injected into rabbits, and the rabbits periodically bledand boosted with the peptide antigen between bleeds. This serum was usedas the source of the antibodies, while the serum before peptideinjection was used as a negative control. The antibodies are affinitypurified by passing the serum over a column composed of the peptide topurify only antibodies that bind the peptide. At least one of theseantibodies in the unpurified state detects a protein of approximatelythe right size that is present in normal plasma but not patient plasma.Antibodies are also prepared against other peptide fragments.

V. Methods of Treatment of TTP

A. Gene Therapy Using ADAMTS13 Coding Sequences

The present invention also provides methods and compositions suitablefor gene therapy to alter ADAMTS13 expression, production, or function.As described above, the present invention provides ADAMTS13 genes andprovides methods of obtaining ADAMTS13 genes from different species.Thus, the methods described below are generally applicable across manyspecies. In some embodiments, it is contemplated that the gene therapyis performed by providing a subject with a wild-type allele of ADAMTS13(i.e., an allele that does not contain a mutation which results in adecrease of VWF-cleaving protease activity; examples of such mutationsare shown in Table 2). Subjects in need of such therapy are identifiedby the methods described above.

Viral vectors commonly used for in vivo or ex vivo targeting and therapyprocedures are DNA-based vectors and retroviral vectors. Methods forconstructing and using viral vectors are known in the art (See e.g.(1992) Miller and Rosman, BioTech., 7:980-990). Preferably, the viralvectors are replication defective, that is, they are unable to replicateautonomously in the target cell. In general, the genome of thereplication defective viral vectors that are used within the scope ofthe present invention lack at least one region that is necessary for thereplication of the virus in the infected cell. These regions can eitherbe eliminated (in whole or in part), or be rendered non-functional byany technique known to a person skilled in the art. These techniquesinclude the total removal, substitution (by other sequences, inparticular by the inserted nucleic acid), partial deletion or additionof one or more bases to an essential (for replication) region. Suchtechniques may be performed in vitro (i.e., on the isolated DNA) or insitu, using the techniques of genetic manipulation or by treatment withmutagenic agents.

Preferably, the replication defective virus retains the sequences of itsgenome that are necessary for encapsidating the viral particles. DNAviral vectors include an attenuated or defective DNA viruses, including,but not limited to, herpes simplex virus (HSV), papillomavirus, EpsteinBarr virus (EBV), adenovirus, adeno-associated virus (AAV), and thelike. Defective viruses, that entirely or almost entirely lack viralgenes, are preferred, as defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Thus, a specific tissue can bespecifically targeted. Examples of particular vectors include, but arenot limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et al.(1991) Mol. Cell. Neurosci., 2:320-330), defective herpes virus vectorlacking a glycoprotein L gene (See e.g., Patent Publication RD 371005A), or other defective herpes virus vectors (See e.g., WO 94/21807; andWO 92/05263); an attenuated adenovirus vector, such as the vectordescribed by Stratford-Perricaudet et al. (J. Clin. Invest., 90:626-630(1992); See also, La Salle et al. (1993) Science 259:988-990); and adefective adeno-associated virus vector (Samulski et al. (1987) J.Virol., 61:3096-3101; Samulski et al. (1989) J. Virol., 63:3822-3828;and Lebkowski et al (1988) Mol. Cell. Biol., 8:3988-3996).

Preferably, for in vivo administration, an appropriate immunosuppressivetreatment is employed in conjunction with the viral vector (e.g.,adenovirus vector), to avoid immuno-deactivation of the viral vector andtransfected cells. For example, immunosuppressive cytokines, such asinterleukin-12 (IL-12), interferon-gamma (IFN-γ), or anti-CD4 antibody,can be administered to block humoral or cellular immune responses to theviral vectors. In addition, it is advantageous to employ a viral vectorthat is engineered to express a minimal number of antigens.

In a preferred embodiment, the vector is an adenovirus vector.Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a nucleic acid of the invention to a variety of celltypes. The present invention contemplates adenoviruses of both human andanimal origin. (See e.g., WO94/26914). Various serotypes of adenovirusexist. Those adenoviruses of animal origin that can be used within thescope of the present invention include adenoviruses of canine, bovine,murine (e.g., Mavl, Beard et al. (1990) Virol., 75-81), ovine, porcine,avian, and simian (e.g., SAV) origin. Preferably, the adenovirus ofanimal origin is a canine adenovirus, more preferably a CAV2 adenovirus(e.g. Manhattan or A26/61 strain (ATCC VR-800)).

Preferably, the replication defective adenoviral vectors of theinvention comprise the ITRs, an encapsidation sequence and the nucleicacid of interest. Still more preferably, at least the E1 region of theadenoviral vector is non-functional. The deletion in the E1 regionpreferably extends from nucleotides 455 to 3329 in the sequence of theAd5 adenovirus (PvuII-BglII fragment) or 382 to 3446 (HinfII-Sau3Afragment). Other regions may also be modified, in particular the E3region (e.g., WO95/02697), the E2 region (e.g., WO94/28938), the E4region (e.g., WO94/28152, WO94/12649 and WO95/02697), or in any of thelate genes L1-L5.

In a preferred embodiment, the adenoviral vector has a deletion in theE1 region (Ad 1.0). Examples of E1-deleted adenoviruses are disclosed inEP 185,573, the contents of which are incorporated herein by reference.In another preferred embodiment, the adenoviral vector has a deletion inthe E1 and E4 regions (Ad 3.0). Examples of E1/E4-deleted adenovirusesare disclosed in WO95/02697 and WO96/22378. In still another preferredembodiment, the adenoviral vector has a deletion in the E1 region intowhich the E4 region and the nucleic acid sequence are inserted.

The replication defective recombinant adenoviruses according to theinvention can be prepared by any technique known to the person skilledin the art (See e.g., Levrero et al. (1991) Gene 101:195; EP 185 573;and Graham (1984) EMBO J., 3:2917). In particular, they can be preparedby homologous recombination between an adenovirus and a plasmid thatcarries, inter alia, the DNA sequence of interest. The homologousrecombination is accomplished following co-transfection of theadenovirus and plasmid into an appropriate cell line. The cell line thatis employed should preferably (i) be transformable by the elements to beused, and (ii) contain the sequences that are able to complement thepart of the genome of the replication defective adenovirus, preferablyin integrated form in order to avoid the risks of recombination.Examples of cell lines that may be used are the human embryonic kidneycell line 293 (Graham et al. (1977) J. Gen. Virol., 36:59), whichcontains the left-hand portion of the genome of an Ad5 adenovirus (12%)integrated into its genome, and cell lines that are able to complementthe E1 and E4 functions, as described in applications WO94/26914 andWO95/02697. Recombinant adenoviruses are recovered and purified usingstandard molecular biological techniques that are well known to one ofordinary skill in the art.

The adeno-associated viruses (AAV) are DNA viruses of relatively smallsize that can integrate, in a stable and site-specific manner, into thegenome of the cells that they infect. They are able to infect a widespectrum of cells without inducing any effects on cellular growth,morphology or differentiation, and they do not appear to be involved inhuman pathologies. The AAV genome has been cloned, sequenced andcharacterized. It encompasses approximately 4700 bases and contains aninverted terminal repeat (ITR) region of approximately 145 bases at eachend, which serves as an origin of replication for the virus. Theremainder of the genome is divided into two essential regions that carrythe encapsidation functions: the left-hand part of the genome, thatcontains the rep gene involved in viral replication and expression ofthe viral genes; and the right-hand part of the genome, that containsthe cap gene encoding the capsid proteins of the virus.

The use of vectors derived from the AAVs for transferring genes in vitroand in vivo has been described (See e.g., WO 91/18088; WO 93/09239; U.S.Pat. Nos. 4,797,368; 5,139,941; and EP 488 528, all of which are hereinincorporated by reference). These publications describe variousAAV-derived constructs in which the rep and/or cap genes are deleted andreplaced by a gene of interest, and the use of these constructs fortransferring the gene of interest in vitro (into cultured cells) or invivo (directly into an organism). The replication defective recombinantAAVs according to the invention can be prepared by co-transfecting aplasmid containing the nucleic acid sequence of interest flanked by twoAAV inverted terminal repeat (ITR) regions, and a plasmid carrying theAAV encapsidation genes (rep and cap genes), into a cell line that isinfected with a human helper virus (for example an adenovirus). The AAVrecombinants that are produced are then purified by standard techniques.

In another embodiment, the gene can be introduced in a retroviral vector(e.g., as described in U.S. Pat. Nos. 5,399,346, 4,650,764, 4,980,289and 5,124,263; all of which are herein incorporated by reference; Mannet al. (1983) Cell 33:153; Markowitz et al. (1988) J. Virol., 62:1120;PCT/US95/14575; EP 453242; EP178220; Bernstein et al. (1985) Genet.Eng., 7:235; McCormick, (1985) BioTechnol., 3:689; WO 95/07358; and Kuoet al, (1993) :845). The retroviruses are integrating viruses thatinfect dividing cells. The retrovirus genome includes two LTRs, anencapsidation sequence and three coding regions (gag, pol and env). Inrecombinant retroviral vectors, the gag, pol and env genes are generallydeleted, in whole or in part, and replaced with a heterologous nucleicacid sequence of interest. These vectors can be constructed fromdifferent types of retrovirus, such as, HIV, MoMuLV (“murine Moloneyleukaemia virus” MSV (“murine Moloney sarcoma virus”), HaSV (“Harveysarcoma virus”); SNV (“spleen necrosis virus”); RSV (“Rous sarcomavirus”) and Friend virus. Defective retroviral vectors are alsodisclosed in WO95/02697.

In general, in order to construct recombinant retroviruses containing anucleic acid sequence, a plasmid is constructed that contains the LTRs,the encapsidation sequence and the coding sequence. This construct isused to transfect a packaging cell line, which cell line is able tosupply in trans the retroviral functions that are deficient in theplasmid. In general, the packaging cell lines are thus able to expressthe gag, pol and env genes. Such packaging cell lines have beendescribed in the prior art, in particular the cell line PA317 (U.S. Pat.No. 4,861,719, herein incorporated by reference), the PsiCRIP cell line(See, WO90/02806), and the GP+envAm-12 cell line (See, WO89/07150). Inaddition, the recombinant retroviral vectors can contain modificationswithin the LTRs for suppressing transcriptional activity as well asextensive encapsidation sequences that may include a part of the gaggene (Bender et al. (1987) Virol., 61:1639). Recombinant retroviralvectors are purified by standard techniques known to those havingordinary skill in the art.

Alternatively, the vector can be introduced in vivo by lipofection. Forthe past decade, there has been increasing use of liposomes forencapsulation and transfection of nucleic acids in vitro. Syntheticcationic lipids designed to limit the difficulties and dangersencountered with liposome mediated transfection can be used to prepareliposomes for in vivo transfection of a gene encoding a marker (Felgneret. al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7417; See also,Mackey, et al. (1988) Proc. Natl. Acad. Sci. USA 85:8027-8031; Ulmer etal. (1993) Science 259:1745-1748). The use of cationic lipids maypromote encapsulation of negatively charged nucleic acids, and alsopromote fusion with negatively charged cell membranes (Felgner andRingold (1989) Science 337:387-388). Particularly useful lipid compoundsand compositions for transfer of nucleic acids are described inWO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127, hereinincorporated by reference.

Other molecules are also useful for facilitating transfection of anucleic acid in vivo, such as a cationic oligopeptide (e.g.,WO95/21931), peptides derived from DNA binding proteins (e.g.,WO96/25508), or a cationic polymer (e.g., WO95/21931).

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Methods for formulating and administering naked DNA tomammalian muscle tissue are disclosed in U.S. Pat. Nos. 5,580,859 and5,589,466, both of which are herein incorporated by reference.

DNA vectors for gene therapy can be introduced into the desired hostcells by methods known in the art, including but not limited totransfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a genegun, or use of a DNA vector transporter (See e.g., Wu et al. (1992) J.Biol. Chem., 267:963; Wu and Wu (1988) J. Biol. Chem., 263:14621; andWilliams et al. (1991) Proc. Natl. Acad. Sci. USA 88:2726).Receptor-mediated DNA delivery approaches can also be used (Curiel etal. (1992) Hum. Gene Ther., 3:147; and Wu & Wu (1987) J. Biol. Chem.,262:4429).

B. Administration of ADAMTS13 Polypeptides

The present invention also provides methods and compositions suitablefor administering ADAMTS13 to a patient suffering from TTP. As describedabove, the present invention provides nucleotides encoding ADAMTS13 andfragments, mutants, variants, and fusions thereof, and methods ofproducing the encoded polypeptides. The methods described below aregenerally applicable across many species.

In some embodiments, the invention provides a composition comprisingpurified ADAMTS13 peptides; in other embodiments, the invention providesa composition comprising purified ADAMTS13 polypeptide fragments,mutants, variants, or fusions, all of which possess the biologicalactivity of ADAMTS13. Fragments, mutants, variants, or fusions may beused as necessary to alter characteristics of ADAMTS13 to improve itsperformance as a therapeutic treatment of TTP. Such characteristicsinclude stability during storage and administration, circulatinghalf-life, levels of activity, substrate specificity, localization to aparticular tissue, and interaction with other molecules, such asreceptors or enzymatic complexes. For example, the protein is preferablyengineered to have a very long circulating half life. Suchcharacteristics can be introduced as described above. The polypeptidescan be produced as described above. The compositions are formulated asdescribed.

In other embodiments, the invention provides a method of treating apatient with TTP disease, which comprises administering atherapeutically effective amount of ADAMTS13 such that symptoms of thedisease are alleviated. As is well known in the medical arts, dosagesfor any one patient depends upon many factors, including the patient'ssize, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andinteraction with other drugs being concurrently administered. Althoughany method of administration is anticipated, as described further below,preferably the polypeptide is administered intravenously.

VI. Drug Screening Using ADAMTS13

The present invention provides methods and compositions for usingADAMTS13 as a target for screening drugs that can alter, for example,VWF-cleaving protease activity and associated symptoms (e.g., TTPdisease). For example, drugs that induce or inhibit VWF-cleavingprotease activity can be identified by screening for compounds thattarget ADAMTS13 or regulate ADAMTS13 gene expression.

The present invention is not limited to a particular mechanism ofaction. Indeed, an understanding of the mechanism of action is notnecessary to practice the present invention. Nevertheless, it iscontemplated that a decrease of VWF-cleaving protease activity leads toan accumulation of hyperactive large VWF multimers which triggerspathologic platelet aggregation and is the direct mechanism responsiblefor TTP. Thus, it is contemplated that drugs which induce VWF-cleavingprotease activity can be used to prevent symptoms of TTP.

Alternatively, it is also contemplated that increased VWF-cleavingprotease activity could also be used in normal individuals as a novelapproach to anticoagulation (preventing abnormal blood clots). Sinceblood clots are at the basis of many important human diseases includingheart attack and stroke, this new insight could be critical to thedevelopment of new pharmaceuticals to treat these very common humandiseases as well as the rare disorder TTP. Such increased VWF-cleavingactivity could be achieved by inducing the enzyme activity as describedabove. Other embodiments contemplate drugs based upon variants of theADAMTS13 protease itself. Such proteases would, for example, beeffective at reducing clots, be easily administered, and have a lifespan of sufficient duration as to treat the disease, but not to causesubsequent harm.

In one screening method, candidate compounds are evaluated for theirability to alter VWF-cleaving protease activity by adding the compoundin the presence of an ADAMTS13 protease to an assay for the VWF-cleavingprotease activity, for example as is described in Example 1B, anddetermining the effects of the compound on the level of proteaseactivity.

In another screening method, variants of ADAMTS13 are evaluated fortheir ability to cleave VWF by adding the variants to an assay for theVWF-cleaving protease activity, for example as is described in Example1B, and determining the level of protease activity of the variant.

Another technique uses ADAMTS13 antibodies, generated as discussedabove. Such antibodies capable of specifically binding to ADAMTS13peptides can be used to detect the presence of any peptide that sharesone or more antigenic determinants of the ADAMTS13 peptide. Suchpeptides can then be evaluated for protease activity as described above.

The present invention contemplates many other means of screeningcompounds. The examples provided above are presented merely toillustrate a range of techniques available. One of ordinary skill in theart will appreciate that many other screening methods can be used.

In particular, the present invention contemplates the use of cell linestransfected with ADAMTS13 and variants thereof for screening compoundsfor activity, and in particular to high throughput screening ofcompounds from combinatorial libraries (e.g., libraries containinggreater than 10⁴ compounds). The cell lines of the present invention canbe used in a variety of screening methods. In some embodiments, thecells can be used in reporter gene assays that monitor cellularresponses at the transcription/translation level. In still furtherembodiments, the cells can be used in cell proliferation assays tomonitor the overall growth/no growth response of cells to externalstimuli.

The cells are useful in reporter gene assays. Reporter gene assaysinvolve the use of host cells transfected with vectors encoding anucleic acid comprising transcriptional control elements of a targetgene (i.e., a gene that controls the biological expression and functionof a disease target) spliced to a coding sequence for a reporter gene.Therefore, activation of the target gene results in activation of thereporter gene product. Examples of reporter genes finding use in thepresent invention include, but are not limited to, chloramphenicoltransferase, alkaline phosphatase, firefly and bacterial luciferases,β-galactosidase, β-lactamase, and green fluorescent protein. Theproduction of these proteins, with the exception of green fluorescentprotein, is detected through the use of chemiluminescent, colorimetric,or bioluminescent products of specific substrates (e.g., X-gal andluciferin). Comparisons between compounds of known and unknownactivities may be conducted as described above.

VII. Pharmaceutical Compositions Containing ADAMTS13 Nucleotides,Peptides, and Antibodies, and Analogs

The present invention further provides pharmaceutical compositions whichmay comprise all or portions of ADAMTS13 encoding polynucleotidesequences, ADAMTS13 polypeptides, inhibitors or antagonists of ADAMTS13bioactivity, including antibodies, alone or in combination with at leastone other agent, such as a stabilizing compound, and may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water.

The methods of the present invention find use in treating diseases oraltering physiological states characterized by decreased VWF-cleavingprotease activity, and/or pathologic platelet aggregation. The inventionprovides methods for increasing VWF-cleaving protease activity and/ordecreasing pathologic platelet aggregation by administering peptides orpeptide fragments or variants of ADAMTS13. Alternatively, drugs whichact to increase VWF-cleaving protease activity and/or decreasingpathologic platelet aggregation, as discovered through screening methodsdescribed above, are administered.

Peptides can be administered to the patient intravenously in apharmaceutically acceptable carrier such as physiological saline.Standard methods for intracellular delivery of peptides can be used(e.g., delivery via liposome). Such methods are well known to those ofordinary skill in the art. The formulations of this invention are usefulfor parenteral administration, such as intravenous, subcutaneous,intramuscular, and intraperitoneal. Therapeutic administration of apolypeptide intracellularly can also be accomplished using gene therapyas described above.

As is well known in the medical arts, dosages for any one patientdepends upon many factors, including the patient's size, body surfacearea, age, the particular compound to be administered, sex, time androute of administration, general health, and interaction with otherdrugs being concurrently administered.

Accordingly, in some embodiments of the present invention, ADATS13nucleotides and ADAMTS13 amino acid sequences can be administered to apatient alone, or in combination with other nucleotide sequences, drugsor hormones or in pharmaceutical compositions where it is mixed withexcipient(s) or other pharmaceutically acceptable carriers. In oneembodiment of the present invention, the pharmaceutically acceptablecarrier is pharmaceutically inert. In another embodiment of the presentinvention, ADAMTS13 encoding polynucleotide sequences or ADAMTS13 aminoacid sequences may be administered alone to individuals subject to orsuffering from a disease, such as TTP or stroke.

Depending on the condition being treated, these pharmaceuticalcompositions may be formulated and administered systemically or locally.Techniques for formulation and administration may be found in the latestedition of “Remington's Pharmaceutical Sciences” (Mack Publishing Co,Easton Pa.). Suitable routes may, for example, include oral ortransmucosal administration; as well as parenteral delivery, includingintramuscular, subcutaneous, intramedullary, intrathecal,intraventricular, intravenous, intraperitoneal, or intranasaladministration.

For injection, the pharmaceutical compositions of the invention may beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. For tissue or cellular administration,penetrants appropriate to the particular barrier to be permeated areused in the formulation. Such penetrants are generally known in the art.

In other embodiments, the pharmaceutical compositions of the presentinvention can be formulated using pharmaceutically acceptable carrierswell known in the art in dosages suitable for oral administration. Suchcarriers enable the pharmaceutical compositions to be formulated astablets, pills, capsules, liquids, gels, syrups, slurries, suspensionsand the like, for oral or nasal ingestion by a patient to be treated.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. For example, aneffective amount of ADAMTS13 may be that amount that results inVWF-cleaving protease activity, or decreased levels of plateletaggregation, comparable to normal individuals who are not suffering fromTTP or stroke. Determination of effective amounts is well within thecapability of those skilled in the art, especially in light of thedisclosure provided herein.

In addition to the active ingredients these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries that facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known (e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes).

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are carbohydrate or protein fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; starch from corn,wheat, rice, potato, etc; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; andgums including arabic and tragacanth; and proteins such as gelatin andcollagen. If desired, disintegrating or solubilizing agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, (i.e., dosage).

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients mixed with a filler orbinders such as lactose or starches, lubricants such as talc ormagnesium stearate, and, optionally, stabilizers. In soft capsules, theactive compounds may be dissolved or suspended in suitable liquids, suchas fatty oils, liquid paraffin, or liquid polyethylene glycol with orwithout stabilizers.

Compositions comprising a compound of the invention formulated in apharmaceutical acceptable carrier may be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. For polynucleotide or amino acid sequences of ADAMTS13,conditions indicated on the label may include treatment of conditionrelated to apoptosis.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-% mannitol at a pH range of 4.5 to 5.5 that is combined with bufferprior to use.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. Then, preferably, dosage can be formulated in animalmodels (particularly murine models) to achieve a desirable circulatingconcentration range that adjusts ADAMTS13 levels.

A therapeutically effective dose refers to that amount of ADAMTS13 orvariant or drug that ameliorates symptoms of the disease state. Toxicityand therapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index, and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. The dataobtained from these cell culture assays and additional animal studiescan be used in formulating a range of dosage for human use. The dosageof such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage varies within this range depending upon the dosage form employed,sensitivity of the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors which may be taken into account include theseverity of the disease state; age, weight, and gender of the patient;diet, time and frequency of administration, drug combination(s),reaction sensitivities, and tolerance/response to therapy. Long actingpharmaceutical compositions might be administered every 3 to 4 days,every week, or once every two weeks depending on half-life and clearancerate of the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature (See, U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212,all of which are herein incorporated by reference). Those skilled in theart will employ different formulations for ADAMTS than for the inducersor enhancers of ADAMTS13. Administration to the bone marrow maynecessitate delivery in a manner different from intravenous injections.

VIII. Transgenic Animals Expressing Exogenous ADAMTS13 Genes andHomologs, Mutants, and Variants Thereof

The present invention contemplates the generation of transgenic animalscomprising an exogenous ADAMTS13 gene or homologs, mutants, or variantsthereof. In preferred embodiments, the transgenic animal displays analtered phenotype as compared to wild-type animals. In some embodiments,the altered phenotype is the overexpression of mRNA for an ADAMTS13 geneas compared to wild-type levels of ADAMTS13 expression. In otherembodiments, the altered phenotype is the decreased expression of mRNAfor an endogenous ADAMTS13 gene as compared to wild-type levels ofendogenous ADAMTS13 expression. In other embodiments, the transgenicmice have a knock out mutation of the ADAMTS13 gene. In still furtherembodiments, the altered phenotype is expression of an ADAMTS13 mutantgene; non-limiting examples of such mutants are shown in Table 1. Inpreferred embodiments, the transgenic animals display a TTP diseasephenotype. Methods for analyzing the presence or absence of such alteredphenotypes include Northern blotting, mRNA protection assays, RT-PCR,detection of protein expression with antibodies, and detection ofprotein activity with VWF-cleaving protease activity, such as isdescribed in Example 1B.

The transgenic animals of the present invention find use in drug andtreatment regime screens. In some embodiments, test compounds (e.g., adrug that is suspected of being useful to treat TTP disease) and controlcompounds (e.g., a placebo) are administered to the transgenic animalsand the control animals and the effects evaluated. The effects of thetest and control compounds on disease symptoms are then assessed.

The transgenic animals can be generated via a variety of methods. Insome embodiments, embryonic cells at various developmental stages areused to introduce transgenes for the production of transgenic animals.Different methods are used depending on the stage of development of theembryonic cell. The zygote is the best target for micro-injection. Inthe mouse, the male pronucleus reaches the size of approximately 20micrometers in diameter which allows reproducible injection of 1-2picoliters (pl) of DNA solution. The use of zygotes as a target for genetransfer has a major advantage in that in most cases the injected DNAwill be incorporated into the host genome before the first cleavage(Brinster et al. (1985) Proc. Natl. Acad. Sci. USA, 82:4438-4442). As aconsequence, all cells of the transgenic non-human animal will carry theincorporated transgene. This will in general also be reflected in theefficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene. U.S. Pat. No.4,873,191 describes a method for the micro-injection of zygotes; thedisclosure of this patent is incorporated herein in its entirety.

In other embodiments, retroviral infection is used to introducetransgenes into a non-human animal. In some embodiments, the retroviralvector is utilized to transfect oocytes by injecting the retroviralvector into the perivitelline space of the oocyte (U.S. Pat. No.6,080,912, incorporated herein by reference). In other embodiments, thedeveloping non-human embryo can be cultured in vitro to the blastocyststage. During this time, the blastomeres can be targets for retroviralinfection (Janenich (1976) Proc. Natl. Acad. Sci. USA, 73: 1260).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Hogan et al. (1986) inManipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). The viral vector system used to introduce thetransgene is typically a replication-defective retrovirus carrying thetransgene (Jahner et al. (1985) Proc. Natl. Acad Sci. USA 82:6927).Transfection is easily and efficiently obtained by culturing theblastomeres on a monolayer of virus-producing cells (Van der Putten,supra; Stewart, et al. (1987) EMBO J., 6:383). Alternatively, infectioncan be performed at a later stage. Virus or virus-producing cells can beinjected into the blastocoele (Jahner et al. (1982) Nature 298:623).Most of the founders will be mosaic for the transgene sinceincorporation occurs only in a subset of cells that form the transgenicanimal. Further, the founder may contain various retroviral insertionsof the transgene at different positions in the genome that generallywill segregate in the offspring. In addition, it is also possible tointroduce transgenes into the germline, albeit with low efficiency, byintrauterine retroviral infection of the midgestation embryo (Jahner etal. (1982) supra). Additional means of using retroviruses or retroviralvectors to create transgenic animals known to the art involves themicro-injection of retroviral particles or mitomycin C-treated cellsproducing retrovirus into the perivitelline space of fertilized eggs orearly embryos (PCT International Application WO 90/08832 (1990), andHaskell and Bowen (1995) Mol. Reprod. Dev., 40:386).

In other embodiments, the transgene is introduced into embryonic stemcells and the transfected stem cells are utilized to form an embryo. EScells are obtained by culturing pre-implantation embryos in vitro underappropriate conditions (Evans et al. (1981) Nature 292:154; Bradley etal. (1984) Nature 309:255; Gossler et al. (1986) Proc. Acad. Sci. USA83:9065; and Robertson et al. (1986) Nature 322:445). Transgenes can beefficiently introduced into the ES cells by DNA transfection by avariety of methods known to the art including calcium phosphateco-precipitation, protoplast or spheroplast fusion, lipofection andDEAE-dextran-mediated transfection. Transgenes may also be introducedinto ES cells by retrovirus-mediated transduction or by micro-injection.Such transfected ES cells can thereafter colonize an embryo followingtheir introduction into the blastocoele of a blastocyst-stage embryo andcontribute to the germ line of the resulting chimeric animal (forreview, See, Jaenisch (1988) Science 240:1468). Prior to theintroduction of transfected ES cells into the blastocoele, thetransfected ES cells may be subjected to various selection protocols toenrich for ES cells which have integrated the transgene assuming thatthe transgene provides a means for such selection. Alternatively, thepolymerase chain reaction may be used to screen for ES cells that haveintegrated the transgene. This technique obviates the need for growth ofthe transfected ES cells under appropriate selective conditions prior totransfer into the blastocoele.

In still other embodiments, homologous recombination is utilizedknock-out gene function or create deletion mutants. Methods forhomologous recombination are described in U.S. Pat. No. 5,614,396,incorporated herein by reference.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: N (normal); M (molar); mM (millimolar); μM(micromolar); mol (moles); mmol (millimoles); gmol (micromoles); nmol(nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg(micrograms); ng (nanograms); l or L (liters); ml or mL (milliliters);μl or μL (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nm (nanometers); DS (dextran sulfate); ° C. (degreesCentigrade); U (units); ADAM (a disintegrin and metalloproteinase); TPP(thrombotic thrombocytopenic purpura); TSP (thrombospondin); vonWildebrandt factor (VWF) and Sigma (Sigma Chemical Co., St. Louis, Mo.).

Example 1 Methods

This example describes the methods used to identify and characterize thegene ADAMTS13.

-   A. Subjects. Patients included in this study were referred for    evaluation of thrombocytopenia, hemolytic anemia, and schistocytes    on blood smear. Probands for the 4 families (A-D) used in the    linkage analysis all had a chronic relapsing course, responded to    plasma infusion, and had the disorder as neonates or had a family    member with such a disorder as a neonate. The additional probands    studied from families E-G exhibited some or all of these features.    Plasma samples were obtained from sodium citrate anticoagulated    blood by centrifugation and saved at −70° C. as previously described    (Tsai, H. M. & Lian, E. C. Y (1998) N. Engl. J. Med. 339,    1585-1594). Mononuclear cells were obtained from heparin    anticoagulated blood by centrifugation on Ficoll-Hypaque, washed and    transformed with Epstein-Barr virus. Informed consent was obtained    from all individuals prior to sample collection following an    Institutional Review Board approved study protocol.-   B. VWF-cleaving protease activity of patient sera. For the    measurement of VWF-cleaving protease activity, guanidine    hydrochloride-treated VWF was used as the substrate. Protease    activity was represented by the optical density of the dimer of the    176 kd fragment generated from the VWF substrate (Tsai, H. M. &    Lian, E. C. Y. (1998) N. Engl. J. Med. 339, 1585-1594) and was    expressed in U/mL, with the activity measured in pooled normal    control plasma defined as 1 U/mL. Each sample was measured on at    least three occasions and the mean of the results is presented.    Assays for inhibitors of VWF-cleaving protease were performed as    described (Tsai et al. (2001) Clin. Lab. 47, 387-392).-   C. Haplotype analysis. A total of 17 markers were used for haplotype    analysis. 13 of these markers were obtained from the comprehensive    genetics maps of Genethon (Dib, C. et al. (1996) Nature 380,    152-154) and Marshfield (Broman, K. W. et al. (1998) Am. J. Hum.    Genet. 63, 861-869), and 4 of these markers were designed from    publicly available sequence repeat information (see Table 3).

TABLE 3 New STS markers. Marker Accession # BAC 5′ primer 3′ primerGL1-3 pending AL157938 5′-gctttgctctcctgagcttc-3′5′-gtggtgcagttcactgtcgt-3′ (SEQ ID NO: 8) (SEQ ID NO: 9) GL2-1 pendingAL160271 5′-gttgcagtgagctgagatcg-3′ 5′-tgcaggggttttatctccta-3′ (SEQ IDNO: 10) (SEQ ID NO: 11) GL3-2 pending AL1601655′-tgggtgacagagcaagactg-3′ 5′-cttgtatccacgcacagagg-3′ (SEQ ID NO: 12)(SEQ ID NO: 13) GL4-1 pending AC002104 5′-agcctgggtgacagagtgag-3′5′-tacaccaattccccaggtgt-3′ (SEQ ID NO: 14) (SEQ ID NO: 15)

-   D. Linkage analysis. A genome-wide linkage screen was performed    using 382 polymorphic microsatellite markers spaced an average of 10    cM (panels 1-27 of the ABI Prism Linkage Mapping Set-MD1 (Applied    Biosystems)). 20 ng of genomic DNA was amplified using AmpliTaq Gold    DNA polymerase (Applied Biosystems). PCR products were run on an ABI    Prism 3700 DNA Analyzer and analyzed using Genescan v3.5NT and    Genotyper v3.6NT. Inspection of the pedigrees indicated an autosomal    recessive mode of inheritance for TTP in this set of families. The    frequency of the disease gene was assumed to be one per ten thousand    chromosomes in the population. Population frequencies of the marker    alleles were estimated from the genotyped individuals. Two-point LOD    scores were calculated using the program MLINK as implemented in the    FASTLINK package, version 3.0 (Schaffer, A. A. et al. (1994) Hum.    Hered. 44, 225-237) using an autosomal recessive model. A second    series of analyses was performed using a codominant model to reflect    the lowered enzyme levels of individuals who were assumed to be    carriers of the disease gene. For the latter analysis, individuals    were classified as affected (those with clinical diagnoses),    carriers (those with protease levels in the range of 0.45-0.68 U/mL)    and unaffected (those with protease levels in the range of 0.8-1.17    U/mL). Penetrance was set at 100% for both models. Multipoint    analyses were performed with the program VITESSE (O'Connell, J. R. &    Weeks, D. E. (1995) Nat. Genet. 11, 402-408), using the same two    disease models and the 5 markers at or flanking the maximum    two-point LOD score. Order and distances between markers were    determined using the ABI Prism Linkage Mapping Set-MD10 map    information.-   E. Sequence analysis. All exons and intron/exon boundaries of the    predicted ADAMTS13 gene were amplified from patient genomic DNA with    the exception of exon 7, which could not be amplified with multiple    primer sets. Intron primers were selected using the Primer3 software    package available online to allow for analysis of exon sequence as    well as flanking donor and acceptor splice sites. (See Table 4 for    primer sequences). 100 ng of genomic DNA was used in a PCR reaction    using either Platinum Taq DNA polymerase (Invitrogen), the Expand    Long-Template DNA polymerase mix (Roche) or the Advantage 2 DNA    polymerase mix (Clontech). PCR products were either purified    directly from the PCR reaction using the Qiaquick PCR purification    kit (Qiagen) or gel-purified from low-melting agarose (Invitrogen)    using the Wizard PCR preps purification kit (Promega). Total    cellular RNA from lymphoblast cell lines was prepared using Trizol    (Invitrogen) and RT-PCR performed using the One-Step RT-PCR kit    (Invitrogen), according to the manufacturer's instructions.    Sequencing reactions were performed by the University of Michigan    DNA Sequencing Core. Selected PCR products were subcloned into a    pCR-TOPO plasmid (Invitrogen) for further sequence analysis.

TABLE 4 Primers used for the amplification of ADAMTS13 exons andintron/exon boundaries Annealing Exon Forward Primer Sequence ReversePrimer Sequence Temp. 1 5′-CCC TGA ACT GCA ACC ATC TT-3′ 5′-CAA ACC CCAAAG CTG ATG TA- 56¹ (SEQ ID NO: 16) 3′ (SEQ ID NO: 17) 2 5′-TCG GTC TCCCCA AGT GTT AG-3′ 5′-AAC AGG GT GAC AGC AGC TT- 56¹ (SEQ ID NO: 18)3′ (SEQ ID NO: 19) 3 5′-TCT AGA ACC ATC GCC CTC TG-3′ 5′-CCG AGC CAT TCTACC TGA GT-3′ 56¹ (SEQ ID NO: 20) (SEQ ID NO: 21) 4 5′-GCC TCT CCA GCTCTT CAC AC-3′ 5′-GCA TTC TGT GAT CCA TGC TG-3′ 56¹ (SEQ ID NO: 22) (SEQID NO: 23) 5-6 5′-ACG GGC TAG TCA TAG GGT TG-3′ 5′-TAC AAG GAC CCA CTGCTT GC-3′ 56¹ (SEQ ID NO: 24) (SEQ ID NO: 25) 7 Not yet available Notyet available 8 5′-CTT CCA AAC GCT TCC ATC CT-3′ 5′-CCC TCC CAG GAC TAGCTA CA-3′ 56² (SEQ ID NO: 26) (SEQ ID NO: 27) 9 5′-TCT GGG AGG GAC AGTTAA GG-3′ 5′-TAC TGG TCC TGC CTC CTG AC-3′ 56¹ (SEQ ID NO: 28) (SEQ IDNO: 29) 10- 5′-GGG ATC CCT ATG GGT GAG TT-3′ 5′-CCT GGT GTG AAC CAC AGATG- 56¹ 11 (SEQ ID NO. 30) 3′ (SEQ ID NO. 31) 12 5′-GCA CTT TTG TCA CCCCAG TT-3′ 5′-CCA GAG CCT GAA CCA CTT TG-3′ 56² (SEQ ID NO: 32) (SEQ IDNO: 33) 13- 5′-CCC AGA TGC AAA GGA TGA AG-3′ 5′-ATC CAG GGC TGA GTG AGTGT- 56¹ 14 (SEQ ID NO: 34) 3′ (SEQ ID NO: 35) 15 5′-TTT TTC CCG ACC AGCTAA GA-3′ 5′-TCA GAA GTG AGG GCA TCT TG- 56¹ (SEQ ID NO: 36) 3′ (SEQ IDNO: 37) 16 5′-CCG GGA AGG AGA GTC ACT G-3′ 5′-CCC TGT AAG TGA CCG CTGA-3′ 60¹ (SEQ ID NO: 38) (SEQ ID NO: 39) 17- 5′-GTG ATT GCT TGC TGA ACGAA-3′ 5-CAG TGT CCT CAC CTG CAG AA-3′ 56¹ 18 (SEQ ID NO: 40) (SEQ ID NO:41) 19 5′-GAA CAC CTG GAG AGG CTA GG-3′ 5-ACT TAC AAC CGC CAG GTG AC-58³ (SEQ ID NO: 42) 3′ (SEQ ID NO: 43) 20 5′-GAA CCT GCT GGC TGA TGAAT-3′ 5′-GGA TGG TGT TCT TGC TCT GG-3′ 56¹ (SEQ ID NO: 44) (SEQ ID NO:45) 21 5′-CAC ACA CGC CAC TTC CTG-3′ 5′-CCA CGT GTT CCC ATA TAG TCT 56¹(SEQ ID NO: 46) G-3′ (SEQ ID NO: 47) 22 5′-CAC AGC TGG TAA GTG GCA GA-3′5′-CAC AGC TGG TAA GTG GCA GA- 60¹ (SEQ ID NO: 48) 3′ (SEQ ID NO: 49) 235′-TCC CAG CTT CCT GTC TCT TC-3′ 5′-TCT CCT GAT TCA GCT TTC CAA- 60¹(SEQ ID NO: 50) 3′ (SEQ ID NO: 51) 24 5′-AGT ACA CGT GGG TGG AGA GG-3′5′-CTT TCA GGG GAC ACG ATG AG- 56¹ (SEQ ID NO: 52) 3′ (SEQ ID NO: 53) 255′-TTA ACT GCC TCC CAG CTT GT-3′ 5′-CTT TGC CAG GGA GAA AGA GG- 56³ (SEQID NO: 54) 3′ (SEQ ID NO: 55) 26- 5′-ACA GGG TCC ACC CCT ACC T-3′ 5′-CCCAGT TCC TTC CAT CTC AG-3′ 56¹ 27 (SEQ ID NO: 56) (SEQ ID NO: 57) 285′-TAT TGA CCA CAG TGC CAT GC-3′ 5′-TGG TGA ATA TGT GGA GGA 56¹ (SEQ IDNO: 58) AGG-3′ (SEQ ID NO: 59) 29 5′-CCT CGG TTT TCT GGG TAG AG-3′5′-CCA TCC TCG GAG TGG AAT C-3′ 56¹ (SEQ ID NO: 60) (SEQ ID NO: 61)¹PCRs done with Platinum Taq DNA polymerase (Invitrogen) ²PCRs done withExpand Long Template DNA polymerase mix (Roche) ³PCRs done withAdvantage 2 DNA polymerase mix (Clontech)

Genomic DNA was obtained from an additional family. Samples from 2affected individuals as well as from the parents and 6 unaffectedsiblings were available for analysis. Amplification and sequenceanalysis of exons 1-6 and 8-29 and the corresponding exon/intronjunctions of the ADAMTS13 gene were performed on genomic DNA from one ofthe probands as described above. As described above, amplification ofexon 7 could not be achieved despite the use of additional primer pairsdesigned from updated draft genomic sequence. Amplification and sequenceanalysis of exon 26, in which a C>T substitution was identified, wasperformed on genomic DNA from all other members of this family.Allele-specific oligonucleotide hybridization was performed as describedabove.

-   F. Allele-specific oligonucleotide hybridization and restriction    digestion. Individual exons were amplified from 92 unrelated control    individuals. For allele-specific oligonucleotide hybridization, PCR    products were spotted onto nitrocellulose membranes using a dot-blot    apparatus (Invitrogen). 15-mer oligonucleotides corresponding to    wild-type or mutant alleles were end-labeled with γ-32P-ATP using T4    polynucleotide kinase (New England Biolabs). Hybridization was    performed in ExpressHyb solution (Clontech) at 37° C. Blots were    washed in 5× SSPE, 0.1% SDS at a temperature determined empirically    for each oligonucleotide. For restriction digests, 10 μl of PCR    product were digested with enzyme (New England Biolabs), according    to the manufacturer's instructions and products analyzed on a 3%    NuSieve GTG agarose (BioWhittaker Molecular Applications), 1%    agarose (Invitrogen) gel.-   G. RT-PCR and Northern blot analysis. RT-PCR analysis was performed    on cDNA obtained from a Multiple Tissue cDNA panel (Clontech) using    primers 5′-CAGTGCAACAACCCCAGAC-3′ (SEQ ID NO. 62) and    5′-GGCACCTGTCCCATACCTG-3′ (SEQ ID NO. 63), which amplify cDNA    nucleotides 1265-1636. A First-Choice Human Northern Blot (Ambion)    was screened with a probe generated by random priming using the    Rediprime II kit (Amersham) of a PCR product amplified from a human    MOLT4 T cell cDNA library (generated as previously described    (Ginsburg, D. et al. (1985) Science 228, 1401-1406) using the above    primers. Hybridization was performed in ExpressHyb solution    (Clontech) according to manufacturer's specifications. The final    wash step was performed in 0.1×SSC, 0.1% SDS at 50° C.-   H. Isolation of cDNA. A human fetal liver cDNA library in 1gt10    (Clontech) was screened with the Northern probe described above. Two    overlapping cDNA clones were obtained, spanning exons 5-14 and 8-20    of the predicted ADAMTS13 cDNA sequence, respectively. Phage DNA was    purified using a Nucleobond lambda midi kit (Clontech), digested    with EcoRI (New England Biolabs) and subcloned into pBSII-SK+    (Stratagene). 5′ RACE was performed on RLM-RACE-ready human liver    cDNA (Ambion) using the following primers:    5′-GTGTCGTCCTCAGGGTTGAT-3′ (SEQ ID NO: 64) (outer) and    5′-GGCTCTGTCAGAATGACCATC-3′ (SEQ ID NO: 65) (inner). Marathon    RACE-ready human liver cDNA (Clontech) was used for 3′ RACE using    primers 5′-TGCCAGGTGGGAGGTGTCAGAG-3′ (SEQ ID NO: 66) (outer) and    5′-GCCTGGCCTTTGAGAACGAGAC-3′ (SEQ ID NO: 67) (inner) and for nested    RT-PCR using primers 5′-CATTGGCGAGAGCTTCATC-3′ (SEQ ID NO: 68) and    5′-ATGGGGAGGGAGCCTTCT-3′ (SEQ ID NO: 69) (outer) and    5′-ACCCTGAGCCTGTGTGTGTC-3′ (SEQ ID NO: 70) and    5′-GCAGAGGTGGCATCCAGA-3′ (SEQ ID NO: 71) (inner) to amplify a    product spanning cDNA nucleotides 1552-2625. RACE and RT-PCR    products were cloned into a pCR-TOPO vector (Invitrogen) and    individual clones were subjected to sequence analysis. Sequence    bridging that obtained by 5′ RACE and that of the overlapping cDNA    clones (cDNA nucleotides 389-534) was obtained from the C9ORF8 EST    cluster (Unigene cluster Hs.149184). Exon-intron boundaries and    sequence accuracy were verified against publicly available draft    human sequence.-   I. Generation of ADAMTS13 mammalian expression construct and    mutants. An ADAMTS13 cDNA encompassing exons 1-29 was assembled and    cloned into the EcoRI and EcoRV sites of pCDNA3.1 (Invitrogen). The    cloned fragment corresponds to nucleotides 62-4390 in the ADAMTS13    cDNA (GenBank accession number AF414401), encompassing the entire    ADAMTS13 coding sequence. The following sequence was inserted into    the EcoRI site of the vector in order to include an optimized Kozak    consensus sequence (Kozak (1991) J. Biol. Chem. 266, 19867-19870)    (uppercase) 5′-tcgatcctcgagtctagaGCCGCCACCATG-3′ (SEQ ID NO. 72),    with the underlined ATG serving as the start codon. Nucleotides    1-707 (with the A of the ATG designated +1) were derived from IMAGE    EST clone 1874472 (GenBank accession number AI281246); nucleotides    708-896, and 897-1748 were derived from two previously described    cDNA clones isolated from a human fetal liver cDNA library    (Clontech), nucleotides 1749-2918 and 2919-4329 were derived from    previously described RT-PCR and 3′ RACE products. An error in the 3′    RACE clone (insertion of a G at position 3631 of AF414401) was    corrected by site-directed mutagenesis using the GeneEditor    mutagenesis system (Promega).

Nine ADAMTS13 missense mutations shown in Table 1 were engineered intothe full-length construct by site-directed mutagenesis using theGeneEditor mutagenesis system (Promega).

A construct encoding a C-terminal epitope tagged version of the ADAMTS13cDNA was engineered by PCR through the replacement of the sequencespanning the ADAMTS13 termination codon to the Not I site of pcDNA3.1 inthe construct above (encoding exons 1-29) with the following sequenceencoding a FLAG epitope (DYKDDDDK) (SEQ ID NO. 73)5′-gactacaaggacgacgacgacaagtaggcggccgc-3′ (SEQ ID NO. 74).

DNA for transfection was prepared using the PerfectPrep plasmid XL(Eppendorf) or Maxi (Qiagen) kits.

-   J. Transfections. Polyoma T-antigen expressing CHO cells (CHO-Tag)    (Smith & Lowe (1994) J. Biol. Chem. 269, 15162-15171) were cultured    in alpha-MEM (supplemented with deoxyribonucleotides and    ribonucleotides), containing 10% heat-inactivated fetal bovine    serum, 0.4 mg/ml G418, penicillin and streptomycin (Life    Technologies). Cells were split into 6-well culture dishes    (Costar, 3516) at 6×105 cells/well 48 hours before transfection.    Transfections were performed in triplicate for each construct. Four    mg of each DNA (pcDNA3.1, pCDNA3.1-ADAMTS13 and pcDNA3.1-ADAMTS13    mutants 1-9) were introduced into the cells using Lipofectamine 2000    (Invitrogen) according to manufacturer's optimized conditions for    CHO-K1 cells. As a transfection control, 25 ng of pSEAP2-Control    vector (BD Biosciences), encoding secreted alkaline phosphatase, was    co-transfected with each DNA. Cells were washed three times with    D-PBS (Life Technologies) and serum-free ∀-MEM was added 18 hours    following transfection. Conditioned media were collected 48 hours    following transfection. One milliliter of conditioned media was    concentrated approximately 20-fold using Ultrapure-30 columns    (Amicon). Secreted alkaline phosphatase activity in 1 ml of    concentrated conditioned media was measured using the Great EscAPe    SEAP detection kit (BD Biosciences) and read in a TD-20 luminometer    (Turner Designs). Volumes of conditioned media were normalized to    the sample with the lowest transfection efficiency and equal volumes    (10 ml) were used for the measurement of VWF-cleaving protease    activity. Secreted alkaline phosphatase activity in conditioned    media from cells transfected with pcDNA3.1 alone was 1.9-43 fold    higher than in media from cells transfected with the various    constructs. The latter controls were thus not normalized for    transfection efficiency (samples were used undiluted) in order to    obtain the most conservative estimate of background VWF-cleaving    protease activity present in conditioned media. When taking into    account the concentration factor for each of the wild-type samples    (8.5-10.2 fold), and the VWF-cleaving protease activity in    conditioned media of cells transfected with wild-type, the ADAMTS13    construct ranged from 4.2-4.7 U/ml, with 1 U/ml representing the    VWF-cleaving protease activity present in pooled normal plasma.-   K. VWF-cleaving protease assay of transfected cells. VWF-cleaving    protease assays were performed as previously described (Tsai et    al. (2001) Clin. Lab 47, 387-392). Assays were performed blindly and    in triplicate for each transfection. The activity in 1 ml of pooled    normal human plasma was designated as 1 U. The lack of activity in    serum-free and serum-replete media was verified. Results shown in    FIG. 11 represent the means of three transfections for each mutant,    with error bars representing standard deviations. Statistical    significance was determined using ANOVA.-   L. Generation of anti-peptide antibodies. Anti-peptide antibodies    against ADAMTS13 were generated and affinity purified by a    commercial supplier (Research Genetics). Antibodies were raised in    rabbits against the following peptides: 1) SQTINPEDDTDPGHAD (SEQ ID    NO: 75) (metalloprotease domain), 2) ESFIMKRGDSFLDGTR (SEQ ID    NO: 76) (cysteine-rich domain), 3) GRLTWRKMCRKLLD (SEQ ID NO: 77)    (CUB domain), and 4) CPEMQDPQSWKGKEGT (SEQ ID NO: 78) (C-terminus).    The cysteine at the N-terminus of the last peptide was artificially    added for conjugation purposes.-   M. Western blot analysis. Conditioned media from CHO-Tag cells    transfected with wild-type and mutant ADAMTS13 constructs, or    mock-transfected with empty pcDNA3.1 vector, were subjected to    SDS-PAGE and transferred to nitrocellulose membranes. Membranes were    blocked in 5% powdered milk in PBS and incubated with anti-peptide    antibodies (1:500) or anti-FLAG M2 monoclonal antibody (Sigma,    1:500). Membranes were then washed in TBS-Tween and incubated with    either HRP-conjugated goat anti-rabbit (Sigma, 1:5000) or HRP    conjugated goat anti-mouse (Sigma, 1:10,000). Chemiluminescent    detection was performed using ECL reagent (Amersham).

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inchemistry, and molecular biology or related fields are intended to bewithin the scope of the following claims.

1. A composition comprising recombinant ADAMTS13 polypeptide that is atleast 95% identical to SEQ ID NO:2 from amino acid position 75 to aminoacid position 1427 having von Willebrand factor (VWF) cleaving proteaseactivity wherein said composition is free of proteins found in humanplasma other than said recombinant ADAMTS13 polypeptide.
 2. Thecomposition of claim 1, wherein said recombinant ADAMTS 13 has an aminoacid sequence that is at least 99% identical to SEQ ID NO:2 from aminoacid position 75 to amino acid position
 1427. 3. The composition ofclaim 1, wherein said recombinant ADAMTS 13 is purified from arecombinant cell culture.
 4. The composition of claim 3, wherein saidrecombinant ADAMTS 13 is purified using chromatography.
 5. Thecomposition of claim 4, wherein the chromatography is selected from thegroup consisting of anion exchange chromatography, cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatography,high performance liquid chromatography and lectin chromatography.
 6. Thecomposition of claim 3, wherein the recombinant ADAMTS 13 is purifiedusing a precipitation and extraction procedure.
 7. A pharmaceuticalcomposition comprising recombinant ADAMTS 13 polypeptide that is atleast 95% identical to SEQ ID NO:2 from amino acid position 75 to aminoacid position 1427 having yon Willebrand factor (VWF) cleaving proteaseactivity, wherein said composition is free of proteins found in humanplasma other than said recombinant ADAMTS 13 , and a pharmaceuticallyacceptable carrier.
 8. The pharmaceutical composition according to claim7, wherein the isolated, recombinant ADAMTS13 has an amino acid sequencethat is at least 99% identical to SEQ ID NO: 2 from amino acid position75 to amino acid position
 1427. 9. The pharmaceutical compositioncomprising according to claim 7, wherein the composition is formulatedin an aqueous solution.
 10. The pharmaceutical composition according toclaim 7, further comprising a drug or a hormone.
 11. The pharmaceuticalcomposition according to claim 7, wherein the composition is formulatedfor systemic administration to a subject.